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* Department of Experimental Pathology, Institute for Medical Sciences, Kyoto University, Kyoto, Japan;
Department of Neurology, Oregon Health and Science University, Portland, OR 97201;
Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR 97201;
L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland; and
¶ Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, but not TNF-
, whereas coculture with IL-18 enhanced the secretion of TNF-, but not INF-. However, coculture with both IL-18 and IL-12 induced high levels of both TNF- and IFN-. Additionally, IL-12 selectively enhanced mRNA expression of CCR5, whereas IL-18 selectively enhanced the expression of CCR4 and CCR7, and CCR4 and CCR5 were coexpressed on the surface of T cells cocultured with IL-12 and IL-18. Finally, estrogen treatment, previously found to inhibit both TNF- and IFN- production, completely abrogated all signs of passive EAE. These data demonstrate that optimal potentiation of encephalitogenic activity can be achieved by conditioning MOG-specific T cells with the combination of IL-12 and IL-18, which, respectively, induce the secretion of IFN-/CCR5 and TNF-/CCR4/CCR7, and that estrogen treatment, which is known to inhibit both proinflammatory cytokines, can completely ablate this aggressive form of passive EAE. |
Introduction |
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, IL-2, TNF-, and lymphotoxin (1, 2). IL-12 and IL-18 are both strong inducers of Th1 cells. IL-12 drives Th1 differentiation directly (3, 4), in part through the induction of IFN-, and IL-18 functions as a cofactor for Th1 development (5, 6) through the induction of TNF- by CD3+/CD4+ and NK cells (7). These two cytokines are known to induce IFN- production by T cells and NK cells synergistically (5, 8, 9). The synergism is mediated by the interaction of two signal transduction molecules, STAT-4 and AP-1 (10), and reciprocal induction of their receptors by either ligand (11, 12). IL-12 and IL-18 have been found to be important factors regulating the induction of EAE. The administration of anti-IL-12 or anti-IL-18 Ab has been reported to suppress the induction of EAE (13, 14). It has also been reported that both IL-12 and IL-18 knockout mice are resistant to the induction of EAE (15, 16).
In this study we employed a passive EAE system in which myelin oligodendrocyte glycoprotein (MOG) peptide-specific T cells were activated in the presence of IL-12 and IL-18. T line cells stimulated with anti-CD3/anti-CD28 and incubated with both cytokines secreted high levels of IFN- and TNF-; up-regulated CCR4, CCR5, and CCR7; and transferred severe clinical and histological signs of EAE into susceptible mice. Moreover, treatment with estrogen, which is known to inhibit the production of both TNF- and IFN-, completely inhibited this aggressive form of passive EAE. This is the first report demonstrating selective induction of TNF- and CCR4/CCR7 by IL-18 and the requirement for both IL-18 and IL-12 coculture for potentiating encephalitogenic activity by MOG-specific T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were housed in the Animal Resource Facility at Portland Veterans Affairs Medical Center in accordance with institutional guidelines.
Antigens
Mouse MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized using solid phase techniques and was purified by HPLC at Beckman Institute, Stanford University (Palo Alto, CA).
MOG35–55-specific T cell lines
To prepare T cell lines specific for MOG35–55 peptide, C57BL/6 mice were immunized s.c. in the flanks with 0.2 ml of an emulsion containing 200 µg of MOG35–55 in saline and an equal volume of CFA containing 400 µg Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI). Ten days after immunization, lymph node cells were cultured with MOG35–55 (20 µg/ml) at 8 x 106 cells/ml in stimulation medium (RPMI 1640 medium supplemented with nonessential amino acids, sodium pyruvate, 2-ME, and 10% FBS) for 48 h. The T cells were expanded in medium containing IL-2 (100 U/ml). After 5–10 days in culture, the T cell lines responded specifically to MOG35–55 peptide.
Induction of EAE
Before passive transfer to induce EAE, MOG35–55-specific T cell lines (1 x 106 cells/ml) were cultured with anti-mouse CD3 (1 µg/ml; clone 145-2C11; BD PharMingen, San Diego, CA) and anti-CD28 (1 µg/ml; clone 37.51; BD PharMingen) Abs immobilized on a tissue culture plate with or without IL-12 (20 ng/ml; R&D Systems, Minneapolis, MN) and/or IL-18 (25 ng/ml; MBL, Nagoya, Japan) for 24 h. After the cells were washed three times with PBS, they were transferred i.p. into naive B6 recipients (1–4 x 106 cells/mouse). On the same day and 2 days after the cell transfer, the mice were injected i.v. with pertussis toxin (67 ng/mouse; List Biological Laboratories, Campbell, CA). The mice were assessed daily for clinical signs of EAE according to the following scale: 0 = normal, 1 = limp tail or mild hindlimb weakness, 2 = moderate hindlimb weakness or mild ataxia, 3 = moderately severe hindlimb weakness, 4 = severe hindlimb 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, moribund condition or dead.
Measurement of cytokine secretion
MOG35–55-specific T cells (1 x 106 cells/ml) were stimulated with anti-mouse CD3/CD28 Ab immobilized on six-well plates in the presence or the absence of IL-12 (20 ng/ml), IL-18 (25 ng/ml), or the combination of IL-12 and IL-18. Cell culture supernatants were recovered 24 h after stimulation and frozen at -70 C until needed for the cytokine assay. Measurement of cytokines was performed by ELISA using cytokine-specific capture and detection Abs (BD PharMingen).
Intracellular staining for cytokines
Cells were cultured at 1–2 x 106 cells/ml in stimulation medium containing 10 µg/ml MOG35–55 peptide. The cells were stimulated for 24 h, the last 5 h in the presence of brefeldin A, washed, and stained with anti-mouse CD4 and/or anti-mouse CD8 CyChrome for 30 min at 4°C. The cells were then washed twice with staining medium (1x PBS, 2% FBS, and 0.02% NaN3), fixed, and permeabilized with Cytofix/Cytoperm solution (BD PharMingen). The cells were then stained with PE-labeled anti-cytokine Abs (anti-mouse TNF- and IFN-) for 30 min at 4°C. Cells were washed twice in perm/wash buffer (BD PharMingen) and once in staining medium before three-color FACS analysis on a FACScan instrument (BD Biosciences, Sunnyvale, CA) using CellQuest software (BD Biosciences). For each experiment cells were stained with isotype control Abs to establish background staining and to set quadrants before calculating the percentage of positive cells.
Extracellular staining for CCRs
MOG35–55-specific T cells (1 x 106 cells/ml) were stimulated with anti-mouse CD3/CD28 (1 µg/ml) Ab immobilized on six-well plates in the presence of IL-12 (20 ng/ml) and IL-18 (25 ng/ml). After 24 h cells were harvested, washed twice with staining media, stained with goat anti-mouse CCR5 and rabbit anti-mouse CCR4 (Santa Cruz Biotechnology, Santa Cruz, CA) for 20 min on ice, washed twice, and stained for 20 min with donkey anti-rabbit F(ab)2 PE (Research Diagnostics, Huntsville, AL) and donkey anti-goat FITC (Santa Cruz Biotechnology). Before flow cytometry (FACScan; BD Biosciences) cells were washed again and resuspended in staining medium. The data were analyzed using CellQuest software (BD Biosciences). For each experiment cells were stained with isotype control Abs to establish background staining and to set quadrants before calculating the percentage of positive cells.
RNase protection assay (RPA)
The same groups of cells used for cytokine evaluation was subjected to CCR mRNA expression. Cells were frozen, subsequently thawed, and evaluated for the expression of CCRs by the RPA. Total RNA was extracted using the STAT-60 reagent (Tel-Test, Friendswood, TX). Chemokine expression was determined using the RiboQuant RPA kit (BD PharMingen) according to the manufacturer’s instructions. The CCR set detected the following transcripts: CCR1, CCR1b, CCR2, CCR3, CCR4, and CCR5. Additionally, the riboprobe for CCR7 was used. The sample loading was normalized by the housekeeping gene L32, which was included in each template set. RPA analysis was performed on 20 µg of total RNA hybridized with probes labeled with [32P]UTP. After digestion of ssRNA, the RNA pellet was solubilized and resolved on a 5% sequencing gel. For quantification, gels were exposed by phosphorimaging (Bio-Rad, Hercules, CA), and radioactivity in individual bands (after background subtraction) in comparison with L32 was assessed with Quantity One software (Bio-Rad).
Histology
Perfused, intact spinal columns were removed from four different groups of mice after adoptive transfer of MOG35–55-specific T cells treated or untreated with IL-12 and/or IL-18 and stimulated with anti-CD3/anti-CD28 Abs. The spinal cords were dissected after fixation in 10% phosphate-buffered formalin and embedded in paraffin before sectioning. The sections were stained with Luxol Fast Blue/periodic acid-Schiff/hematoxylin and analyzed by light microscopy.
Estrogen treatment
Sixty-day release pellets containing 15 or 2.5 mg of 17
-estradiol (E2; Innovative Research of America, Sarasota, FL) were implanted s.c. in the scapular region behind the neck using a 12-gauge trochar as described by the manufacturer (Innovative Research of America). The mice were implanted 2 wk before the induction of passive EAE with the E2 pellets. The concentrations of E2 expected in the serum induced by 15- and 2.5-mg E2 pellets were 5,000–10,000 and 1,500–2,000 pg/ml, respectively, which represent levels found during pregnancy and 5 times less than the level found during pregnancy. E2 levels measured previously (17) were equivalent to those reported by the manufacturer.
Statistical analyses
Significant differences in incidence and mortality between control and E2-treated mice were assessed using Fisher’s exact test. Differences in the day of disease onset, cytokine production, and mRNA expression were determined using Student’s t test. Differences in the peak score and cumulative disease index (CDI), which was calculated over 40 days after adoptive transfer, were assessed by Mann-Whitney U test and/or ANOVA. A value of p 
0.05 was considered significant.
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Results |
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In this study we used MOG35–55-specific T cell lines, which were generated from C57BL/6 mice by immunization with MOG35–55 peptide in CFA followed by restimulation of primed lymph node cells in vitro with autoantigen for 2 days. All MOG35–55-specific T cell lines responded specifically to MOG35–55 as measured by proliferation (Fig. 1). Before additional stimulation with MOG35–55 or anti-CD3 and anti-CD28 mAbs, the T line cells were expanded in IL-2 for 5–10 days. MOG35–55-stimulated T cells acquired only mild encephalitogenic activity, with transfer of 4–10 x 106 cells inducing maximum EAE scores of 0–1 (data not shown). For subsequent studies we used T cells activated with anti-CD3/CD28 Abs alone (control) or with the addition of IL-12 and/or IL-18. As shown in Fig. 2, A and B, transfer of 4 million MOG35–55-specific T cells activated with anti-CD3/CD28 mAbs in the absence of IL-12 or IL-18 failed to transfer EAE. However, in the presence of IL-12, these T cells induced moderate EAE with delayed onset in 
60% of recipients. Similar transfer of MOG35–55-specific T cells activated in the presence of IL-18 induced mild EAE in only one of five mice (Fig. 2, A and B). In contrast, transfer of 4 million MOG35–35-specific T cells activated with anti-CD3/CD28 mAbs in the presence of both IL-12 and IL-18 induced severe and chronic EAE in all recipient mice, and doses even as low as 1 million T cells retained the ability to induce EAE (data not shown). Generally, histological EAE mirrored clinical disease, with increased perivascular inflammatory lesions in mice with more severe EAE (Fig. 3). Mice developing severe disease after transfer of T cells activated in the presence of IL-12 and IL-18 had mononuclear cell infiltrates in the CNS composed of 45% CD4+, 30% CD8+, and 25% MAC-1+ cells (data not shown).
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and IFN- production by MOG35–55-specific T cells
It has been reported that the encephalitogenicity of myelin-specific CD4+ T cell clones correlated with production of TNF- (1). In addition, an MOG89–101-specific T cell hybridoma transduced with TNF- was found to exacerbate EAE (18). Moreover, CD4+ T cells infiltrating into the CNS exclusively produced IFN- (19). Therefore, we determined levels of secreted cytokines from MOG35–55-specific T cells after stimulation without or with IL-12 and/or IL-18 on anti-CD3/CD28 Ab-coated plates (Table I). After stimulation of T cell lines with anti-CD3/CD28 Abs in the absence of cytokines, consistent levels of TNF- and IFN- were induced. However, in the presence of IL-18, but not IL-12, the T cell lines secreted nearly 2.5-fold higher levels of TNF-, and this effect was not diminished when both IL-18 and IL-12 were added. In contrast, IFN- production was mainly enhanced by IL-12, which induced 3-fold higher levels of IFN- than controls, compared with only a modest 37% increase with IL-18. However, the presence of both IL-12 and IL-18 produced a synergistic effect on IFN- secretion (Table I). It is noteworthy that MOG35–55-specific T cells, upon stimulation with MOG35–55 peptide (after expanding in IL-2), secreted 10 times less TNF- and 26 times less IFN- compared with T cells stimulated with CD3/CD28 Abs only (data not shown).
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and TNF-, T line cells were activated in the presence or the absence of IL-12, IL-18, or both factors and stained for intracellular expression. The MOG35–55-specific T cell line, which contained 30% CD4+ and 60% CD8+ T cells, stained 99% positive for intracellular IFN- and 65% positive for intracellular TNF- after stimulation with anti-CD3/CD28 Abs. Incubation with IL-12, IL-18, or both factors did not significantly change the percentage of either CD4+ or CD8+ T cells expressing IFN- or TNF- in the cell line. IL-12 and IL-18 differentially up-regulate CCR4, CCR5, and CCR7 expression, and IL-12/IL-18-conditioned cells coexpress extracellular CCR4 and CCR5
Since trafficking of encephalitogenic and recruited T cells into CNS is highly dependent on CCR expression, we were interested in the CCR profile of MOG-specific T cells used in our study as a function of IL-12 and/or IL-18 costimulation and encephalitogenic activity. We found that the up-regulation of CCRs was very selective for each cytokine. As shown in Fig. 4B and quantified in Fig. 4A, IL-12 selectively up-regulated CCR5 expression, whereas IL-18 selectively up-regulated CCR4 expression (p < 0.05), while simultaneously down-regulating the expression of the other CCRs. Interestingly, the message was enhanced for both CCR4 and CCR5 in T cells incubated with IL-12 and IL-18. To further demonstrate cell surface expression of CCRs on IL-12- and IL-18-conditioned cells, we assessed extracellular staining by flow cytometry. As shown in Fig. 5, essentially all stained MOG35–55-specific T cells (9%) after conditioning with IL-12/IL-18 coexpressed both CCR4 and CCR5. Of note MOG35–55-specific T cells, after stimulation with MOG35–55 peptide, displayed message for CCR2 and CCR5, but no message for CCR4 and only weak message for CCR1 (data not shown).
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0.05) up-regulated by treatment with IL-18 compared with control cells stimulated only with anti-CD3/CD28 or IL-12-treated cells (Fig. 6). The increase in CCR7 expression remained when the T cells were stimulated with both IL-12 and IL-18, although the magnitude of the change was reduced (Fig. 6).
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Our prior work established that estrogen (E2) has potent inhibitory effects on intracellular production of both TNF- and IFN-, and we sought to evaluate E2 effects on EAE induced by cytokine-activated T cell lines. Two weeks before passive transfer of IL-12 and IL-18 culture-enhanced MOG35–55-specific T cell lines, mice were implanted with 2.5- or 15-mg E2 pellets, which maintain a constant serum E2 levels of 1,500–2,000 and 5,000–10,000 pg/ml, respectively. Our previous studies established that 2.5 mg and higher doses of E2 pellets were very effective in suppressing active EAE induced in C57BL/6 mice by immunization with MOG35–55 peptide in CFA (20, 21). As shown in Table II, the higher 15-mg dose of E2 was very effective at inhibiting EAE induced by adoptive transfer of 4 million IL-12- and IL-18-coactivated MOG35–55-specific T cells, as shown by the lower incidence, delayed onset, and reduced peak and CDI scores compared with vehicle-treated mice. The lower 2.5-mg dose of E2 caused delayed onset of EAE induced by 4, 2, or 1 million transferred T cells, but did not inhibit the incidence or severity of the disease, although there was a strong tendency toward inhibition of the lower disease severity induced by the lowest dose of 1 million T cells. These results indicate that the severe EAE induced with IL-12- and IL-18-enhanced MOG35–55-reactive T cells required a higher concentration of E2 than that needed to inhibit actively induced EAE. In an additional experiment, in vitro treatment with E2 (2,000 pg/ml) of the MOG35–55 peptide-specific T cell line stimulated in the presence of IL-12 and IL-18 nominally reduced intracellular expression of TNF- from 59 to 50% in CD4+ T cells, but had no effect on intracellular TNF- in CD8+ T cells. Neither IL-12 nor IL-18 affected the nearly uniform expression of intracellular IFN- (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CCR5, whereas activation in the presence of IL-18 resulted in up-regulation of TNF-, CCR4, and CCR7. In each case, the T cells transferred only mild clinical and histological EAE. Strikingly, activation in the presence of IL-12 and IL-18 induced both IFN- and TNF-, as well as CCR4, CCR5, and CCR7. This resulting combination resulted in potent encephalitogenic and inflammatory activity of the MOG-specific T cell lines that could only be reversed by E2 concentrations 6-fold higher than that needed to inhibit actively induced EAE. Taken together, these findings strongly underscore the differential potentiating effects of IL-12 and IL-18 and the need for preconditioning with both cytokines to induce full encephalitogenic activity.
Although IL-12 enhanced the secretion of IFN-, it did not affect the already high percentage (98%) of T cells with intracellular IFN-. Similarly, treatment with IL-18 increased the secretion of TNF-, but had little effect on the percentage (65%) of T cells with intracellular TNF-. These data indicate that IL-12 and IL-18 enhanced the amounts of IFN- and TNF- produced per cell, and that the same cells were producing both cytokines.
IL-18 and IL-12 are known to be potent inducers of Th1 immune responses. The critical role of IL-12 was shown in IL12p40 knockout mice, in which T cells failed to produce IFN- (22). IL-18 possesses proinflammatory properties by stimulating macrophage inflammatory protein-1, monocyte chemoattractant protein-1, and TNF- from CD3+ CD4+ T cells and NK cells, with subsequent induction of IL-8 and IL-1 from the CD14+ population (7). We have further observed that IL-18 transgenic mice have an enhanced number of TNF-+ CD4+ T cells, but not CD11b+ cells, in spleen (unpublished observations). On the other hand, IL-18-deficient mice are defective in mounting autoreactive Th1 cells and autoantibody responses and are resistant to MOG35–55-induced EAE (16). Fully differentiated wild-type Th1 cells after BCG infection did not produce augmented levels of IFN- by in vitro stimulation with IL-18 alone. However, these cells secreted high levels of IFN- in response to IL-12 or when stimulated by the combination of IL-12 and IL-18 (6). Although each cytokine displays its own pattern of action, recent studies stress that the key event in mounting an effective immune response is the cooperation of these two important cytokines. The IL-18 in synergy with IL-12 facilitates priming of Th1 immune responses (23, 24, 25) and production of IFN- by many cell types, including T cells (11, 26, 27), NK cells (6), B cells (28), dendritic cells (29, 30), and macrophages (31, 32). In mice lacking both cytokines, NK activity and Th1 cell differentiation were drastically decreased (6).
In addition to up-regulating IFN- and TNF-, IL-12 and IL-18 induced selective expression of CCR5 and CCR4/CCR7, respectively. MOG35–55-specific T cells pretreated with both cytokines had high mRNA expression of CCR4, CCR5, and CCR7, and FACS analysis revealed coexpression of CCR4 and CCR5 on all stained T cells (9%). CCR5 belongs to the family of inflammatory CCRs up-regulated upon T cell activation and is primarily expressed on memory cells (33, 34, 35). CCR5 expression is required for cells to move from secondary lymphoid tissue to the inflamed sites in the periphery. Up-regulation of CCR5 expression on T and B cells in accordance with an increased expression of their ligands, macrophage inflammatory protein-1, and RANTES was found in multiple sclerosis and EAE (36, 37, 38) and was found to be diminished after different EAE treatment strategies (37, 39). In our studies IL-12 alone, but not IL-18, was a potent up-regulator of CCR5 expression on MOG-specific T cells. Selective up-regulation of CCR5 by IL-12 has been observed by others (40, 41), although not on encephalitogenic T cells.
In contrast, MOG-specific T cells treated with IL-18 showed significant down-regulation of CCRs typically found in spinal cords of sick animals, including CCR1, CCR2, and CCR5 (37), but a strong up-regulation of CCR4 and CCR7. To the best of our knowledge, this is the first study showing up-regulation of either CCR4 or CCR7 by IL-18. CCR7 was initially implicated in lymphocyte homing to secondary lymphoid tissue in response to the specific chemokine ligands, CCL19 and CCL21 (42), and CCR7 ligand has been implicated in homeostatic expansion of autoreactive T cells (43). Recently, CCR7 was found on encephalitogenic T cells, and CCL19 and CCL21 were found on inflamed venules in the brains of mice with EAE (44). Taken together, these findings suggest that up-regulation of CCR7 by IL-18 is an important indicator of disease-inducing capacity by MOG-specific T cells.
The exact role for CCR4 is still unknown. CCR4 is expressed by circulating memory CLA+ lymphocytes, and its ligand thymusand activation-regulated chemokine is expressed on normal and inflamed cutaneous endothelium (45). However, CCR4 was expressed in the CLA-47 subset of peripheral blood memory CD4+ lymphocytes (46). Both CCR5 and CCR4 were found to be critical CCRs in rheumatoid joints (47). The CCR4 expression significantly correlated with the severity of active systemic lupus erythematosus (48). Recently it was documented that the binding of Ag-primed T cells to DC was mediated by CCR4 (49). Our results suggest a strong correlation between simultaneous expression of CCR4, CCR5, and CCR7 and the ability of MOG T cells to acquire potent encephalitogenic activity.
Finally, our study demonstrated that 6-fold higher doses of estrogen were required to inhibit passive EAE induced by IL-12- and IL-18-activated T cells than were needed to inhibit actively induced EAE with MOG35–55 peptide/CFA. Th1-immune responses in females are more vigorous than those in males, making females more susceptible to autoimmune diseases. One of the major contributors in responsiveness to self-Ags is sex hormones (50). It is well established that pregnancy levels of estrogen are immunosuppressive and can strongly diminish disease symptoms in several autoimmune diseases, including multiple sclerosis and its animal model, EAE. We have previously demonstrated that treatment with low levels of both E2 and estriol reduced the severity of EAE caused by active immunization (17, 51), mainly by reducing the expression of TNF- and, to a lesser degree, IFN- by T cells, macrophages, dendritic cells, and microglial cells (20, 21, 52). The high dose of E2 required to inhibit passive EAE in this report is indicative of the strong degree of coactivation induced by the combination of IL-12 and IL-18. Although the low dose of E2 that normally would inhibit active EAE failed to inhibit passive disease under the conditions employed, this treatment did cause delayed onset of EAE and had a marked tendency to diminish the clinical severity of EAE induced by the lowest dose of transferred T cells. Consistent with our previous unpublished data, in vitro treatment of IL-12- and IL-18-activated T cells with E2 had little effect on T cell activation, although a nominal reduction in intracellular TNF- was noted in our current experiments.
In conclusion, we found that 1) incubation of MOG35–55-specific T cells with both IL-12 and IL-18 resulted in potent encephalitogenic activity; 2) encephalitogenic T cells activated in the presence of IL-12 and IL-18 produced high levels of IFN- and TNF-, respectively; 3) activation with IL-12 and IL-18 strongly up-regulated the expression of CCR5 and CCR4/CCR7, respectively; and 4) E2 treatment of severe EAE induced by passive transfer of IL-12- and IL-18-coactivated T cells required 6-fold more estrogen than actively induced EAE.
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Footnotes |
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2 A.I. and A.M. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Halina Offner, Portland Veterans Affairs Medical Center, Neuroimmunology Research R&D-31, 3710 SW U.S. Veterans Hospital Road, Portland, OR 97239. E-mail address: offnerva{at}ohsu.edu
4 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CDI, cumulative disease index; E2, 17-estradiol; MOG, myelin oligodendrocyte glycoprotein; RPA, RNase protection assay.
Received for publication July 24, 2002. Accepted for publication February 20, 2003.
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H. Kato, A. Ito, J. Kawanokuchi, S. Jin, T. Mizuno, K. Ojika, R. Ueda, and A. Suzumura Pituitary adenylate cyclase-activating polypeptide (PACAP) ameliorates experimental autoimmune encephalomyelitis by suppressing the functions of antigen presenting cells Multiple Sclerosis, December 1, 2004; 10(6): 651 - 659. [Abstract] [PDF]
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L. Garidou, S. Laffont, V. Douin-Echinard, C. Coureau, A. Krust, P. Chambon, and J.-C. Guery Estrogen Receptor {alpha} Signaling in Inflammatory Leukocytes Is Dispensable for 17{beta}-Estradiol-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis J. Immunol., August 15, 2004; 173(4): 2435 - 2442. [Abstract] [Full Text] [PDF]
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J. Huan, S. Subramanian, R. Jones, C. Rich, J. Link, J. Mooney, D. N. Bourdette, A. A. Vandenbark, G. G. Burrows, and H. Offner Monomeric Recombinant TCR Ligand Reduces Relapse Rate and Severity of Experimental Autoimmune Encephalomyelitis in SJL/J Mice through Cytokine Switch J. Immunol., April 1, 2004; 172(7): 4556 - 4566. [Abstract] [Full Text] [PDF]
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), IL-18 (
), or the combination of IL-12 and IL-18 (
) on anti-CD3- and anti-CD28-coated plates were transferred i.p. to C57BL/6 mice. The data are expressed as the mean clinical score for all animals as a function of days after transfer of MOG35–55-specific T cells and are combined results from more than two experiments.
