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* Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR 97239;
Department of Neurology, Oregon Health & Science University, Portland, OR 97239;
Department of Anesthesiology and Perioperative Medicine, Oregon Health & Science University, Portland, OR 97239;
Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR 97239; and
¶ Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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1, or on FoxP3 that is expressed by regulatory T cells. Moreover, pretreatment of PLP-139–151-specific T cells with RTL401 in vitro induced high levels of secreted IL-13, with lesser induction of other pro- and anti-inflammatory cytokines. Given the importance of IL-13 for protection against EAE, these data strongly implicate IL-13 as a dominant regulatory cytokine induced by RTL therapy. Pronounced IL-13 levels coupled with marked reduction in IL-6 levels secreted by PLP-specific T cells from blood after treatment of mice with RTL401 indicate that IL-13 and IL-6 may be useful markers for following effects of RTL therapy in future clinical trials in multiple sclerosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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2 MHC domain (6), and were found to inhibit T cell activation and prevent experimental autoimmune encephalomyelitis (EAE) in rodents (3, 7, 8). In our previous studies, we designed and tested RTLs that included the minimal TCR interface, which involves only the 
1 and 1 MHC domains covalently linked to peptide without CD4 binding (9). These constructs signaled directly through the TCR as a partial agonist (10), prevented and treated myelin basic protein-induced monophasic EAE in Lewis rats (11, 12), inhibited activation but induced IL-10 secretion in human DR2-restricted T cell clones specific for myelin basic protein-85–99 or cABL peptides (13, 14), and reversed chronic clinical and histological EAE induced by myelin oligodendrocyte glycoprotein-35–55 peptide in DR2 transgenic mice (15) in an MHC- and Ag-specific manner. We further developed a monomeric RTL for use in SJL mice that develop a relapsing form of EAE after injection with proteolipid protein (PLP)-139–151 peptide in CFA (16). This RTL, comprised of an I-As/PLP-139–151 peptide construct (RTL401), prevented relapses and reversed clinical and histological EAE through a mechanism involving cytokine switching that differed strikingly from our previous studies using rat and human RTLs in other models of EAE (16).
The cytokine milieu in SJL/J mice or other rodent strains developing actively induced EAE is strongly affected by the use of the highly inflammatory CFA. Patients with multiple sclerosis (MS) have increased levels of high avidity Th1 cells specific for neuroantigens (17), but these T cells do not arise by planned immunization with CFA or other strong adjuvants. We reasoned that RTL401 treatment of active EAE and, more specifically, the RTL401-induced effect on encephalitogenic T cells might be influenced by this strong proinflammatory environment. Thus, we evaluated the inhibitory mechanisms of RTL401 therapy of EAE induced passively by transfer of activated T cells from PLP-139–151 peptide-immunized donors to naive SJL/J recipient mice. RTL401 injected i.v. or s.c. reversed clinical severity of passive EAE and reduced, but did not prevent cellular infiltration into the CNS in a manner similar to its ability to treat active EAE. Moreover, RTL401 therapy prevented injury to axons during EAE. Interestingly, in the absence of CFA, RTL401 treatment strongly enhanced production of the Th2 cytokine, IL-13, in spleen, blood, and spinal cord tissue, with variable effects on other Th1 and Th2 cytokines, and no significant effect on the Th3 cytokine, TGF-1, or on FoxP3 that is expressed by regulatory T (Treg) cells. Moreover, pretreatment of PLP-139–151-specific T cells with RTL401 in vitro induced high levels of secreted IL-13, with lesser induction of other pro- and anti-inflammatory cytokines. These are the first data demonstrating that the therapeutic mechanism of RTL401 on EAE involves induction of IL-13 and prevention of axonal injury.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Female SJL mice were obtained from The Jackson Laboratory at 7–8 wk of age. The mice were housed at the animal facility at Portland Veterans Affairs Medical Center in accordance with institutional guidelines.
RTL construction and production
General methods for the design, cloning, and expression of RTLs have been described previously (9, 11, 14). In brief, mRNA was isolated from the splenocytes of SJL mice using an Oligotex Direct mRNA mini kit (Qiagen). cDNA of the Ag binding/TCR recognition domain of murine I-As MHC class II was derived from mRNA using two pairs of PCR primers. The two chains were sequentially linked by a 5-aa linker (GGQDD) in a two-step PCR with NcoI and XhoI restriction sites being added to the N terminus of the 1 chain and to the C terminus of the 1 chain, respectively, to create RTL400. The PLP-139–151 peptide with a linker (GGGGSLVPRGSGGGG) was covalently linked to the 5' end of the 1 domain of RTL400 to form RTL401. The murine I-As 11 insert was then ligated into pET21d+ vector and transformed into Nova blue Escherichia coli host (Novagen) for positive colony selection and sequence verification. RTL400 and RTL401 plasmid constructs were then transformed into E. coli strain BL21(DE3) expression host (Novagen). The purification of proteins has been described previously (14). The final yield of purified protein varied between 15 and 30 mg/L bacterial culture.
Dynamic light-scattering analysis
Light-scattering experiments were conducted in a DynaPro molecular sizing instrument (Protein Solutions). The protein samples, in 20 mM Tris-Cl buffer at pH 8.5, were filtered through 100-nm Anodisc membrane filters (Whatman) at a concentration of 1.0 mg/ml, and 20 µl of filtered sample was loaded into a quartz cuvette and analyzed with a 488-nm laser beam. Fifty spectra were collected at 4°C to get an estimation of the diffusion coefficient and relative polydispersity of the protein in aqueous solution. Data were then analyzed with Dynamics software V.5.25.44 (Protein Solutions), and buffer baselines were subtracted. Data were expressed as the mean hydrodynamic radius of the protein sample in nm. The m.w. of the RTLs was estimated with Dynamics software V.5.25.44 (Protein Solutions).
Circular dichroism (CD) analysis
CD analyses were performed, as previously described (14), using an Aviv Model 215 CD spectrometer (Aviv Associates), except that the recombinant proteins were in Tris-Cl buffer at pH 8.5. Spectra were averaged and smoothed using built-in algorithms with buffer baselines subtracted. Secondary structure was estimated using a built-in deconvolution software package (CDNN version 2.1) and the Variable Selection method.
Organ stimulation, cell transfer, and RTL treatment
SJL mice were immunized with 150 µg of PLP-139–151 (C140S) in 200 µg of CFA. Ten days postimmunization, lymph nodes and spleens were harvested and cultured in vitro in the presence of 10 µg/ml PLP-139–151 peptide in stimulation medium containing 2% FBS for 48 h. Cells were then washed, and 15 million blasting cells were injected i.p. into SJL mice. The mice were assessed daily for signs of EAE according to the following scale: 0 = normal; 1 = limp tail or mild hind limb weakness; 2 = moderate hind limb weakness or mild ataxia; 3 = moderately severe hind limb weakness; 4 = severe hind limb weakness or mild forelimb weakness or moderate ataxia; 5 = paraplegia with no more than moderate forelimb weakness; and 6 = paraplegia with severe forelimb weakness or severe ataxia or moribund condition. The cumulative disease index (CDI) is the sum of the daily EAE scores for each mouse for the entire duration of the experiment. The CDI is presented as mean ± SD for each group. At the onset of clinical signs of EAE, the mice were divided into three groups and treated as controls or with 100 µl of 1 mg/ml RTL401 i.v. along with antihistamine for 5 days or RTL401 s.c for 8 days. Mice were monitored for disease until they were sacrificed for ex vivo analyses.
Histopathology
Intact spinal cords were removed from mice on day 19 of clinical disease and fixed in 10% Formalin. The spinal cords were dissected after fixation and embedded in paraffin before sectioning. The sections were stained with H&E to assess inflammatory lesions, and analyzed by light microscopy. Semiquantitative analysis of inflammation was determined by examining at least 10 replicates of the cervical, thoracic, and lumbar sections from each mouse.
Western blot (immunoblotting) detection of nonphosphorylated neurofilaments (NPNFL)
The procedure was conducted, as described by Pitt et al. (18). PBS-perfused spinal cords were homogenized in ice-cold RIPA+ buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM NaCO3) and protease inhibitors and incubated for 15 min with shaking. After centrifugation (14,000 x g at 4°C for 15 min), the supernatant was collected and the protein concentration was measured and adjusted using RIPA+ buffer. Samples were denatured in sampling buffer for 10 min at 70°C, then separated by 10% SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. After transfer, the membrane was blocked for 1 h in 3% BSA. Immunodetection was accomplished by incubation overnight at 4°C with primary mAb SMI 32 (1/5,000 dilution in 3% BSA and 0.05% Tween 20; purchased from Sternberger Monoclonals) specific for NPNFL. After being washed, the blots were incubated with HRP-labeled goat Ab against mouse IgG (1/5,000 dilution in 3% BSA and 0.05% Tween 20; purchased from Pierce) for 1 h and then washed. Blots were developed with a SuperSignal West Pico Chemiluminescent kit (Pierce). To precisely control the amounts of protein loaded, the membranes were stripped with the Restore Western Blot Stripping Buffer (Pierce) and detected again with a mAb for GAPDH purchased from Chemicon International. After being developed, the films were scanned and quantified with ImageQuant software (Amersham Biosciences).
Cytokine determination by cytometric bead array (CBA)
Brains were pooled from three mice from each group and processed through a fine mesh screen. The mononuclear cells were then isolated on a 40–80% Percoll gradient, and 1 x 106 brain cells were cultured along with 3 x 106 irradiated splenocytes (used as filler cells) in a 24-well plate in the presence of 10 µg/ml PLP-139–151 peptide for 48 h. Spleen and blood mononuclear cells were cultured from separate mice at 4 x 106 cells/well in a 24-well flat-bottom culture plate in stimulation medium with 10 µg/ml PLP-139–151 peptide for 48 h. Supernatants were then harvested and stored at –80°C until tested for cytokines. The mouse inflammation CBA kit was used to detect IL-12p40, TNF-, IFN-
, MCP-1, IL-10, and IL-6 simultaneously (BD Biosciences). Briefly, 50 µl of sample was mixed with 50 µl of the mixed capture beads and 50 µl of the mouse PE detection reagent. The tubes were incubated at room temperature for 2 h in the dark, followed by a wash step. The samples were then resuspended in 300 µl of wash buffer before acquisition on the FACScan. The data were analyzed using the CBA software (BD Biosciences). Standard curves were generated for each cytokine using the mixed bead standard provided in the kit, and the concentration of cytokine in the supernatant was determined by interpolation from the appropriate standard curve.
ELISA for detection of IL-13 and IL-4
Spleens, blood, and brains from control, RTL i.v., and RTL s.c. mice were harvested on day 19 postimmunization, and 4 x 106 cells were cultured in stimulation medium in the presence of 10 µg/ml PLP-139–151 for 48 h. For the in vitro assays, cells were cultured in the presence of APC with or without PLP-139–151 (2 µg/ml). Supernatants were harvested and frozen at –80°C until further testing. Ninety-six-well plates were coated with 100 µl of anti-mouse IL-13 or IL-4 capture Ab (4 µg/ml) in 1x PBS or sodium bicarbonate coating buffer. Plates were incubated at 4°C overnight. Plates were then washed with wash buffer (1x PBS/0.05% Tween 20) and blocked with blocking buffer (1x PBS, 2% BSA) for 2 h at room temperature. Plates were then washed, and 100 µl of sample or standard was added to each well. IL-13 plates were incubated at room temperature for 2 h, while IL-4 plates were incubated at 4°C overnight. The following day, plates were washed and 100 µl of biotinylated Ab (IL-13 or IL-4) was added. IL-13 plates were incubated at room temperature for 2 h, while IL-4 plates were incubated at room temperature for 45 min. Plates were then washed, and 100 µl of 1/200 diluted HRP was added to IL-13 plates and 1/400 diluted HRP was added to the IL-4 plates. Plates were incubated at room temperature for 30 min, followed by a wash step. This was followed by addition of 100 µl of tetramethylbenzidine chromogen (Kirkegaard & Perry Laboratories catalogue no. 52-00-2). The plates were allowed to develop for 
30 min, and reaction was stopped by adding 100 µl of stop solution (Kirkegaard & Perry Laboratories catalogue no. 50-85-05). The OD was then measured at 450 nm.
RNA isolation and RT-PCR
Total RNA was isolated from spinal cords using the RNeasy mini kit protocol (Qiagen) and then converted to cDNA using oligo(dT), random hexamers, and Superscript RT II enzyme (Invitrogen Life Technologies). Real-time PCR was performed using Quantitect SYBR Green PCR master mix (Qiagen) and primers (synthesized by Applied Biosystems). Reactions were conducted on the ABI Prism 7000 Sequence Detection System (Applied Biosystems) to detect the following genes: L32 (F, GGA AAC CCA GAG GCA TTG AC; R, TCA GGA TCT GGC CCT TGA AC); IFN- (F, TGC TGA TGG GAG GAG ATG TCT; R, TGC TGT CTG GCC TGC TGT TA); TNF- (F, CAG CCG ATG GGT TGT ACC TT; R, GGC AGC CTT GTC CCT TGA); IL-10 (F, GAT GCC CCA GGC AGA GAA; R, CAC CCA GGG AAT TCA AAT GC); TGF-1 (F, CCG CTT CTG CTC CCA CTC; R, GGT ACC TCC CCC TGG CTT); TGF-3 (F, GGG ACA GAT CTT GAG CAA GC; R, TGC AGC CTT CCT CCC TCT C); IL-13 (F, ACT GCT CAG CTA CAC AAA GCA ACT; R, TGA GAT GCC CAG GGA TGG T); IL-4 (F, GGA GAT GGA TGT GCC AAA CG; R, CGA GCT CAC TCT CTG TGG TGT T); FoxP3 (F, GGC CCT TCT CCA GGA CAG A; R, GCT GAT CAT GGC TGG GTT GT).
Statistical analysis
Statistical difference between vehicle and treatment groups was determined by the Mann-Whitney U test. Differences in cytokine levels were evaluated by Student’s t test. A p value 
0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In a previous study, we demonstrated that an RTL containing the 1 and 1 domains of the I-As class II molecule covalently linked to the encephalitogenic PLP-139–151 peptide (termed RTL401) could reverse clinical signs in SJL/J mice with actively induced relapsing EAE (16). This effect was specific, because treatment of SJL mice with a different RTL containing DR2/myelin oligodendrocyte glycoprotein-35–55 peptide did not affect the course of EAE. The use of CFA during active induction of EAE causes systemic inflammatory changes that may exaggerate cytokine profiles associated with the pathogenic mechanism and obfuscate changes induced by RTL therapy. Thus, in the current study, we evaluated the therapeutic effects of RTL401 on passive EAE induced in SJL mice by injection with 15 million activated PLP-139–151-specific T cells.
At onset of clinical signs of EAE (usually day 6 after transfer), the mice were treated with vehicle or RTL401 i.v. for 5 days or s.c. for 8 days. Both the i.v. and s.c. routes of administration were very effective at stopping disease progression and reversing clinical signs of disease throughout the observation period ending on day 19 after passive transfer of T cells (Fig. 1A). The vehicle-treated mice (n = 8) showed a CDI of 46 ± 10.5, whereas the i.v.-treated mice (n = 8) had a CDI of 19.5 ± 5.1 compared with 21.4 ± 9.9 in the s.c.-treated mice (n = 8, p < 0.01 for both routes). The peak disease score was also significantly lower for both i.v.- and s.c.-treated mice (4.5 ± 0.9 for vehicle vs 2.3 ± 1.0 for s.c group vs 2.1 ± 0.4 for the i.v group, p < 0.01), representing only a minimal progression of EAE in the RTL401-treated group before sustained reduction in clinical scores. The striking therapeutic effect of RTL401 was highly reproducible in a second experiment (CDI of 50.5 ± 4.4 for the vehicle group vs 18.9 ± 7.9 for the RTL i.v.-treated mice, n = 7 for each group, p < 0.01; Fig. 1B). The peak intensity of disease was also markedly suppressed following RTL i.v. treatment (4.9 ± 0.2 in control- vs 2.4 ± 0.8 in RTL-treated mice).
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Histopathological examination of spinal cords taken on day 19 from vehicle-treated mice showed inflammatory lesions with dense and focal mononuclear infiltrates (Fig. 2A). In contrast, there was a marked reduction of these lesions in day 19 spinal cords of RTL401-treated mice (Fig. 2B). Treatment with RTL401 also resulted in a 60% reduction in recovered mononuclear cells from brain tissue (2 x 106 from vehicle vs 8 x 105 from RTL i.v.).
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Relapsing and progressive EAE results in axonal injury similar to that observed in MS. To evaluate the effects of RTL therapy on axonal survival during EAE, we assessed NPNFL by Western blots in spinal cords of RTL401- and vehicle-treated mice (i.v. route) on day 19 after T cell transfer. At onset of EAE when treatment began (day 6), the signal intensities of staining for NPNFL and the control marker, GAPDH, were unchanged in mice with a clinical score of 2.0 relative to asymptomatic naive mice (Fig. 3). However, at the completion of treatment on day 19, vehicle-treated mice with a clinical score of 4.0 had a 60% increase in staining for NPNFL compared with naive or pretreated mice or RTL401-treated mice with a clinical score of 1.5 that showed no evidence of axonal injury (Fig. 3). The results from this and two repeat experiments indicated that early treatment with RTL401 preserved neurofilaments and prevented further axonal injury due to progression of EAE.
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Spleen, blood, and brain were harvested from control-, RTL i.v.-, and RTL s.c.-treated mice on day 19. Mononuclear cells were isolated and then cultured in the presence of 10 µg/ml PLP-139–151 peptide for 48 h, and the culture supernatants were then assayed for the level of secreted cytokines. In splenocytes from vehicle-treated mice with EAE, the predominant cytokines induced by the PLP-139–151 peptide were IFN- and IL-13. Interestingly, treatment with RTL401 i.v. and s.c. induced significant increases in the production of both Th1 (TNF-, IFN-, IL-6; Fig. 4A) and Th2 cytokines (IL-13, IL-4, IL-10; Fig. 4B).
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and IL-13 production (Fig. 4). TNF- and IL-10 levels were low initially and did not change after treatment with RTL401.
In brain mononuclear cells from mice with EAE, as in spleen, IFN- and IL-13 were the predominant cytokines induced by the PLP-139–151 peptide (Fig. 4). In contrast, treatment with RTL401 i.v. and s.c. had a strong suppressive impact on both pro- and anti-inflammatory responses in the brain (Fig. 4), possibly due to the decrease in the number of infiltrating lymphocytes.
mRNA expression in spleen and spinal cord of vehicle- vs RTL401-treated mice
To further evaluate cytokine expression profiles, we evaluated mRNA levels in spleen and spinal cord tissue (without further stimulation with PLP-139–151 peptide) from vehicle- vs RTL401 i.v.-treated mice with EAE. In general, changes in splenic cytokine mRNA levels induced by RTL401 treatment reflected changes in secreted protein levels. There was a significant increase in proinflammatory cytokines, IFN- and TNF-, but also a marked increase in Th2 cytokines (IL-4 and IL-13), the Tr1 cytokine (IL-10), and TGF-3, which we previously associated with protection against EAE (19) in splenocytes from RTL401 i.v.-treated mice (Fig. 5). However, no significant changes were observed in the expression of the Treg cell marker, FoxP3, or TGF-1 in spleen (Fig. 5), suggesting that Treg cells and Th3 cells may not be involved in the RTL treatment mechanism. These changes were highly representative of data averaged from three separate experiments shown in Table I. In spinal cord tissue, mRNA expression was increased 6-fold for IL-13 and 2-fold for IFN- (Fig. 6 and Table I), but otherwise, cytokine expression was reduced or unchanged. Notably, FoxP3 expression was strongly reduced in spinal cord tissue from treated mice, suggesting that RTL therapy prevented further recruitment of Treg cells into the CNS.
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To evaluate how RTLs affect T cell responses in vitro, we incubated PLP-139–151 peptide-specific T cells used in the passive transfer experiments with 100 or 10 µg/ml RTL401 for 24 h before the addition of irradiated splenocyte APCs and further incubation for 48 h to assess cytokine secretion profiles. As controls, we preincubated the T cells with medium or 10 µg/ml free PLP-139–151 peptide, which represents the molar equivalent of peptide contained in the 100 µg/ml dose of the RTL401 construct. As is shown in Fig. 7, PLP-139–151-specific T cells preincubated with medium produced negligible levels (<50 pg/ml) of both inflammatory (TNF-, IFN-, and IL-6) and noninflammatory (IL-13, IL-10, and IL-4) cytokines. T cells preincubated with free PLP-139–151 peptide had substantial increases in secretion of all cytokines, particularly IL-13 (1,000 pg/ml) and, to a lesser extent, TNF- (400 pg/ml). Of importance, preincubation of T cells with 100 µg/ml RL401 (neat) produced a striking increase in secretion of all of the cytokines, again with a predominant effect on IL-13 (12,000 pg/ml, a 12-fold increase) and a lesser effect on TNF- (3,000 pg/ml, a 7.5-fold increase). Preincubation of the T cells with 10 µg/ml RTL401 (1:10) produced cytokine responses similar to 10 µg/ml free PLP peptide, even though the concentration of bound peptide in the RTL401 preparation was only 1 µg/ml. These results clearly demonstrate that preincubation of PLP-specific T cells with RTL401 before addition of APC, but without additional peptide, induces significantly greater cytokine secretion than the molar equivalent of PLP peptide, resulting in predominant secretion of the Th2 cytokine, IL-13.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Of mechanistic importance, we report for the first time the strong and persistent induction of IL-13 by RTL-targeted T cells obtained from the spleen, blood, and CNS. Moreover, preincubation with RTL401 in vitro primed PLP-139–151-specific T cells to secrete exceedingly high levels (>10 µg/ml) of IL-13 upon addition of APC, without further exposure to PLP-139–151 peptide. Interestingly, RTL401 treatment enhanced the parallel secretion of lesser amounts of IFN-, with variable production of other cytokines. These results further support a mechanism in which RTL therapy induces a cytokine switch in targeted T cells, thus reprogramming pathogenic T cells to produce anti-inflammatory cytokines that help to reduce inflammation in the CNS. We also address for the first time the effect of RTL therapy on axonal injury, a recently described feature of MS (20) that also occurs during the course of EAE (21, 22, 23). Our data demonstrate that treatment with RTL401 at onset of EAE can completely prevent formation of NPNFL, an indicator of axonal injury in CNS, that markedly increased over a 2-wk period in vehicle-treated mice with EAE. The protective effect of RTL401 therapy on axonal survival has also been demonstrated in the active model of EAE (C. Wang, personal communication).
The strong induction of IL-13 by RTL401 may explain a number of observations related to therapy of EAE in SJL/J mice. IL-13 is an important regulatory cytokine in EAE, as demonstrated by Ab reversal of the EAE-protective function of a PLP-139–151-reactive T cell clone stimulated with an altered peptide ligand (24). It is secreted by activated Th2 cells and is known to possess regulatory functions as well as to mediate the pathogenesis of allergic inflammation. It shares many properties with IL-4, owing to the common expression of the IL-4 subunit in their respective receptors (25). Unlike the IL-4R, the IL-13R is expressed on many immune and tissue cells, including B cells, basophils, eosinophils, mast cells, endothelial cells, fibroblasts, monocytes, macrophages, respiratory epithelial cells, and smooth muscle cells (25), but not on T cells (26). This receptor distribution promotes class switching to IgG4 and IgE and promotes hypersensitivity, a possible side effect in SJL/J mice of multiple i.v. injections of RTL401, for which we routinely administer antihistamines (16). However, it also precludes a direct IL-13 regulatory effect on pathogenic Th1 cells in EAE. Alternatively, IL-13 has been shown to inhibit the production of proinflammatory factors produced by monocytes and macrophages, including cytokines (IL-1, IL-6, IL-8, TNF-, and IL-12 (27), but not IFN-), reactive oxygen and nitrogen intermediates, and PGs (25). The cytokine profile of PLP-139–151-reactive mononuclear cells in blood after RTL401 treatment recapitulates this effect, with strongly enhanced levels of IL-13 in combination with a marked decrease in IL-6, a highly inflammatory cytokine known to be essential for induction of EAE (28). In contrast, RTL401 treatment induced a relatively modest increase in IFN-, and minor or no changes in TNF-, IL-4, and IL-10 in blood.
In our previous study, we evaluated cytokine changes induced by RTL401 in spleen, brain, and spinal cord at various times during the course of actively induced EAE (16). During the peak of the initial episode of EAE (day 15), after only three daily injections of RTL401, PLP-139–151-specific splenocytes showed enhanced secretion of TNF-, IFN-, IL-6, and IL-10 in mice that showed clinical improvement. By later time points on day 22 (beginning of the first relapse), and day 28 (peak of first relapse), the splenocyte cytokine profile in RTL401-treated mice was strikingly different, with reduced levels of IFN-, no changes in TNF- or IL-6, and increased levels of IL-10. Subsequently, we demonstrated that low basal levels of IL-13 were not increased in RTL401-treated mice with actively induced EAE at any time point, indicating that IL-13 expression may be obfuscated by the strong inflammatory effects of CFA. In contrast, splenocytes from RTL401-treated mice with passive EAE on day 19 (13 days after onset of EAE and initiation of therapy) showed elevated levels of both proinflammatory (TNF-, IFN-, and IL-6) and anti-inflammatory (IL-13, IL-4, and IL-10) cytokines, a profile closely matching that observed in RTL401-treated mice at the peak of the first episode (day 15) of active EAE, but not at later time points (16).
It is potentially important that vehicle-treated mice undergoing passive EAE did not experience distinct relapses (see Fig. 1), suggesting that there was not a natural progression to the relapsing phase induced by active immunization. This difference was most likely reflected by the fact that there were few similarities in cytokine message profiles observed in spinal cord tissue taken on day 19 from RTL401-treated mice with passive EAE (enhanced levels of IL-13 and IFN-) vs spinal cord tissue taken at any time point from RTL401-treated mice with active EAE (decreased levels of inflammatory cytokines, but enhanced levels of Th2-associated CCR3 (29) and TGF-3 that we showed previously to be associated with protection against EAE (19)). Expression of other regulatory molecules, including Th3-associated TGF-1 (30) and Treg-associated FoxP3 (31), was not elevated in RTL401-treated mice with passive EAE. Taken together, it seems likely that the protective mechanisms involved in the treatment of passive EAE included a strong Th2 component (up-regulated IL-4 and especially IL-13 in spleen; up-regulated IL-13 in blood and spinal cord), as well as enhanced expression of TGF-3, and possibly the consistently enhanced levels of IFN- that can be protective under certain conditions (32, 33). IL-10 was also elevated in the spleen of RTL401-treated mice with passive EAE, and it is noteworthy that this cytokine can potentially inhibit EAE if present during the activation or recovery phases of disease (34, 35, 36).
In conclusion, our results for the first time strongly implicate IL-13 as an RTL-induced, Th2-associated cytokine that may play an important regulatory role in reversal of clinical signs in the SJL/J mouse model of EAE. IL-13 can be induced by activation with glatiramer acetate (GA), and it is noteworthy that increased levels of IL-13 and IL-5 were found in the serum of clinical GA responders, but not controls, untreated MS patients, or clinical GA nonresponders (37). The pronounced enhancement of IL-13 coupled with a marked reduction in IL-6 in RTL-targeted blood cells indicate that these cytokines may be useful markers for following effects of RTL therapy in future clinical trials in MS.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants NS47661, NS23444, AI43960, NS41965, and NS46877; National Multiple Sclerosis Society Grants RG3468A and PP1104; Artielle ImmunoTherapeutics; Nancy Davis Multiple Sclerosis Center Without Walls; and the Biomedical Laboratory R&D Service, Department of Veterans Affairs. 
2 Address correspondence and reprint requests to Dr. Halina Offner, Neuroimmunology Research R&D-31, Portland Veterans Affairs Medical Center, 3710 SW U.S. Veterans Hospital Road, Portland, OR 97239. E-mail address: offnerva{at}ohsu.edu
3 Abbreviations used in this paper: RTL, rTCR ligand; CBA, cytometric bead array; CD, circular dichroism; CDI, cumulative disease index; EAE, experimental autoimmune encephalomyelitis; GA, glatiramer acetate; MS, multiple sclerosis; NPNFL, nonphosphorylated neurofilament; PLP, proteolipid protein; Treg, regulatory T.
Received for publication April 6, 2005. Accepted for publication June 29, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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3 is associated with protection against experimental autoimmune encephalomyelitis. Cytokine 25:45.-51.
from an altered peptide ligand-specific Th2 cell clone down-regulate adoptive transfer of experimental autoimmune encephalomyelitis. J. Immunol. 164:3563.-3572. plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.-3227. [Abstract]
confers resistance to experimental allergic encephalomyelitis. Eur. J. Immunol. 26:1641.-1646. [Medline]
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 S. Sinha, S. Subramanian, T. M. Proctor, L. J. Kaler, M. Grafe, R. Dahan, J. Huan, A. A. Vandenbark, G. G. Burrows, and H. Offner A Promising Therapeutic Approach for Multiple Sclerosis: Recombinant T-Cell Receptor Ligands Modulate Experimental Autoimmune Encephalomyelitis by Reducing Interleukin-17 Production and Inhibiting Migration of Encephalitogenic Cells into the CNS J. Neurosci., November 14, 2007; 27(46): 12531 - 12539. [Abstract] [Full Text] [PDF]
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 G. Adamus, G. G. Burrows, A. A. Vandenbark, and H. Offner Treatment of Autoimmune Anterior Uveitis with Recombinant TCR Ligands. Invest. Ophthalmol. Vis. Sci., June 1, 2006; 47(6): 2555 - 2561. [Abstract] [Full Text] [PDF]
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