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The Journal of Immunology, 2004, 173: 6465-6471.
Copyright © 2004 by The American Association of Immunologists

Suppression of Ongoing Experimental Autoimmune Encephalomyelitis by Neutralizing the Function of the p28 Subunit of IL-271

Ruth Goldberg*,{ddagger}, Yaniv Zohar*, Gizi Wildbaum*, Yifat Geron*, Gila Maor{ddagger} and Nathan Karin2,*,{dagger}

* Department of Immunology, {dagger} Rappaport Family Institute for Research in the Medical Sciences, and {ddagger} Department of Morphological Sciences, Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-27 is a recently defined family member of the long-chain, four-helix bundle cytokines, which consist of EBI3, an IL-12p40-related protein, and p28, an IL-12p35-related polypeptide. The role of IL-27 in the regulation of experimental autoimmune encephalomyelitis has never been studied. We show in this study that neutralizing the in vivo function of IL-27 by Abs against IL-27 p28 rapidly suppressed an ongoing long-lasting disease in C57BL/6 mice. These Abs were then used to determine the mechanistic basis of disease suppression. We show in this study that IL-27 is involved not only in the polarization of naive T cells undergoing Ag-specific T cell activation, but also in promoting the proliferation and IFN-{gamma} production by polarized T cells, including the long term Th1 line that has been previously selected against the target encephalitogenic determinant. This may explain in part why neutralizing IL-27 suppresses an already established disease in a very rapid and significant manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune encephalomyelitis (EAE)3 is an autoimmune disease of the CNS that serves as a model for the human disease, multiple sclerosis (MS). In both diseases circulating leukocytes penetrate the blood-brain barrier and damage myelin, resulting in impaired nerve conduction and paralysis (1, 2). Ag-specific T cells direct the initiation and progression of disease. Depending on their cytokine profile, CD4+ T cells fall into different subsets, including Th1 cells that produce large amounts of IFN-{gamma} and TNF-{alpha} and low levels of IL-4; Th2 cells that mostly produce IL-4, IL-5, and IL-13 and, to a much lesser extent, IFN-{gamma} and TNF-{alpha} (3, 4); Th3 cells that produce high levels of TGF-{beta} and, to a much lesser extent, other cytokines (5, 6); Tr1 cells that produce high levels of IL-10 (7); and CD4+CD25+ suppressor T cells (8). The pivotal role of Th1 cells in the initiation and progression of the inflammatory process in several autoimmune diseases, including EAE, has been well documented. Thus, neutralization of IL-12, IL-18, or IFN-{gamma}-inducible protein-10 (CXCL10) suppresses experimentally induced EAE (9, 10, 11) and other T cell-mediated autoimmune diseases (12, 13, 14, 15), while shifting Ag-specific T cell polarization from Th1 to Th2.

IL-27 is a newly defined family member of the long-chain, four-helix bundle cytokines (16). It is a heterodimeric cytokine that consists of EBI3, an IL-12p40-related protein, and p28, an IL-12p35-related polypeptide (16). This cytokine is an early product of activated APCs and drives rapid clonal expansion of naive CD4+ T cells. IL-27 synergizes with IL-12 to trigger IFN-{gamma} production of naive CD4+ T cells and mediates its biologic effects through the orphan cytokine receptor WSX-1/TCCR (16, 17, 18, 19, 20). WSX-1 signaling induces the induction of T-bet through activation of STAT1 during initial Th1 commitment (21, 22, 23). It is therefore possible, although has yet to be proven, that neutralization of IL-27 may affect the regulation of Th1-mediated inflammatory diseases. Very recently, its expression was identified in tissue samples from patients suffering from granulomatous diseases (24). In a very recent study we have demonstrated the ability of neutralizing Abs to IL-27 to suppress adjuvant-induced arthritis (25). However, the role of IL-27 in the regulation of other inflammatory autoimmune diseases, particularity EAE has never been elucidated.

Our laboratory was the first to use the DNA vaccination technology and neutralizing Abs generated by this technology for exploring the roles of different cytokines, chemokines, and Fas ligand in the regulation of EAE and other T cell-mediated autoimmune diseases (10, 14, 26, 27, 28, 29, 30, 31, 32, 33). In this study we use, once again, this powerful technology to obtain highly specific neutralizing Abs to the p28 subunit of IL-27 and to explore the role of this newly defined cytokine in the regulation of EAE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Lewis rats and C57BL/6 mice, ~6 wk old, were purchased from Harlan (Jerusalem, Israel) and maintained under specific pathogen-free conditions in our animal facility.

Peptide Ags

Myelin oligodendrocyte glycoprotein (MOG) p33–55 was ordered from the PAN facility of the Beckman Center of Stanford University. After purification by HPLC, sequence was confirmed by amino acid analysis, and the correct mass was checked by mass spectroscopy. Purification of the peptide that was used in the current study was >95%.

Immunizations and active disease induction

EAE was induced by immunizing mice with MOGp33–55/CFA as described by Mendel et al. (34). Animals were then monitored for clinical signs daily by an observer blind to the treatment protocol. EAE was scored as follows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis; 3, total hind limb paralysis, accompanied by an apparent front limb paralysis; and 4, total hind limb and front limb paralysis.

Induction of transferred EAE

Transferred EAE was inducted by transferring 5–10 x 106 spleen T cells after in vitro priming with MOGp35–55, according to the protocol previously described (35).

Cloning of mouse IL-27 p28, IL-1{beta}, and IL-18

Specific oligonucleotide primers were designed based on its published sequence of each of the above cytokines: mouse IL-27 p28: sense, 5'-ATGGGCCAGGTGACAGGAGACCTTGGCT-3'; antisense, 5'-TTAGGAATCCCAGGCTGAGCCTGGGGCGC-3'; mouse IL-18: sense, 5'-ATGGCTGCCATGTCAGAAGA-3'; antisense, 5'-CTAACTTTGATGTAAGTTAGT-3'; and IL-1{beta}: sense, 5'-ATGGCAACTGTTCCTGAACTCAACTG-3'; antisense, 5'-TTAGGAAGACACTCTTTCCATGGTGAA.

PCR products were cloned into a pUC57/T vector (T-cloning kit K1212; MBI Fermentas, Vilnius, Lithuania) and transformed into Escherichia coli according to the manufacturer’s protocol. Each clone was then sequenced (Sequenase version 2; Upstate Biotechnology, Cleveland, OH) according to the manufacturer’s protocol. PCR products were selected to be used as constructs for naked DNA vaccination only after cloning and sequence verification.

Generation of DNA vaccination-based Abs

The sequenced PCR product of mouse IL-27 p28, IL-18, and IL-1{beta} were transferred into a pcDNA3 vector (Invitrogen Life Technologies, San Diego, CA). Large-scale preparation of plasmid DNA was conducted using Mega Prep (Qiagen, Chatsworth, CA). Four days after immunization, rats were injected into the tibialis anterior muscle with 100 µg of the pcDNA3 vector encoding each of the above cytokines. Four or 5 days after vaccination, one rat from each group was killed, and RT-PCR was applied to tibialis anterior muscle samples. Thereafter, naked DNA vaccine was administered three additional times with intervals of 1–2 wk between each injection. Ten days after the last administration, animals were injected with 100 µg of IL-18 or IL-1{beta} (R&D Systems, Minneapolis, MN) or our rIL-27p28 emulsified in IFA. Ten days later, anti-IL-27, anti-IL-18, or anti-IL-1{beta} Abs were purified from sera of these rats by two steps of purification. A High-Trap Protein G column (BD Biosciences, Piscataway, NJ) was used to purify the IgG fraction. Then cytokine-specific Abs were purified using a cyanogen bromide-activated Sepharose column, as follows. Each recombinant mouse cytokine (5 mg), obtained as described bellow, was bound to cyanogen bromide-activated Sepharose column according to the manufacturer’s instructions (catalogue no. 17-0820-01; Pharmacia Biotech, Uppsala, Sweden). IL-27 p28-specific Abs from sera (IgG fraction) of DNA-vaccinated rats were loaded on the column and then eluted by an acidic elution buffer (glycine, pH 2.5). Isotype determination of the purified Abs (ELISA) revealed that purified Abs were mostly constructed of the IgG2a isotype (data not shown).

Production and purification of rIL-27 p28

PCR product was recloned into a pQE expression vector, expressed in E. coli (Qiagen) and then purified by an Ni-NTA superflow affinity purification of 6xHis proteins (Qiagen). After purification, the purity of rIL-27p28 was verified by gel electrophoresis, followed by sequencing (N terminus) by our sequencing services unit.

Cytokine determination in cultured primary spleen cells

The protein levels of various cytokines were determined using semi-ELISA kits as follows: IFN-{gamma} (murine IFN-{gamma} EliPair; Diaclone. Besançon, France), TNF-{alpha} (murine TNF-{alpha} module set; Bender Medical Systems, Vienna, Austria), IL-4 (murine IL-4 kit, EliPair; Diaclone), and IL-10 ((murine IL-10, EliPair; Diaclone).

FACS analysis

FACS analysis was conducted according to the basic protocol we used previously (30, 36). Before being subjected to intracellular staining, cells were suspended with PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO), ionomycin (0.2 mM; Sigma-Aldrich), and monensin (0.2 mM; Sigma-Aldrich) for 5 h. For intracellular staining, PE-labeled anti-mouse IFN-{gamma} mAb (BD Biosciences, Mountain View, CA) and FITC-labeled anti-mouse IL-4 mAb (BD Pharmingen, San Diego, CA) were used. Cells were analyzed using a FACSCalibur (BD Biosciences). Data were collected for 10,000 events and were analyzed using a CellQuest program (BD Biosciences).

Western blot analysis

Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF-{alpha} (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel. IgG from IL-27 p28 DNA-vaccinated rats or IgG from normal rat serum (final dilution of 1/500 each) were used as primary Abs. Goat anti-rat biotin-conjugated Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary Ab, followed by streptavidin-HRP (Jackson ImmunoResearch Laboratories). The Western blotting Luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA) was then used as a substrate.

Selection of MOG-specific T cell lines

The MOG-specific T cell line was selected according to the protocol described previously (37) with the minor modification of using Con A supernatant as a source of IL-2.

Spot ELISA

Spot ELISAs were conducted according to the detailed protocol described by Min et al. (38). In brief, hemagglutinin multiscreen plates (Millipore, Bedford, MA) were coated with anti-mouse IFN-{gamma} capture Ab (catalogue no. MAB785; R&D Systems) at a concentration of 5 µg/ml in PBS overnight at 4°C. Free sites were saturated with 5% BSA in PBS. Subsequently, 107 spleen cells or line T cells were added. After 24 h of incubation, plates were supplemented with a biotinylated anti-IFN-{gamma} mAb (catalogue no. BAF485; R&D Systems), followed by streptavidin conjugated to avidin-peroxidase (Jackson ImmunoResearch Laboratories). Spots were then counted and measured under a dissecting microscope.

Histopathology

Histological examination of H&E-stained sections of formalin-fixed, paraffin-embedded sections of the lower thoracic and lumbar regions of the spinal cord was performed. Each section was evaluated without knowledge of the treatment status of the animal. The following scale was used: 0, no mononuclear cell infiltration; 1, one to five perivascular lesions per section with minimal parenchymal infiltration; 2, five to 10 perivascular lesions per section with parenchymal infiltration; and 3, >10 perivascular lesions per section with extensive parenchymal infiltration. The mean histological score ± SE was calculated for each treatment group.

Statistical analysis

Significance of differences was examined using Student’s t test. A value of p < 0.05 was considered significant. The Mann-Whitney sum of ranks test was used to evaluate significance of differences in mean maximal clinical scores. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-IL-27 p28 Abs suppress ongoing EAE

A DNA vaccination-based anti-IL-27 p28 polyclonal Ab was constructed as described in Materials and Methods. First, we verified that this Ab is specific to its target. Fig. 1 shows that our Ab binds recombinant mouse IL-27 p28 (lane 1; 27 kDa) and not recombinant mouse IL-18, IL-12, or TNF-{alpha} (lanes 2, 3, and 4, respectively). We also verified (by ELISA) that this Ab does not bind different type I cytokines, including IL-2, IL-4, and IL-15 (data not shown), nor did it bind recombinant soluble {beta}-actin that we constructed under conditions identical with those used to generate rIL-27 (10), but it did bind a 27-kDa band in supernatant of activated primary cultured lymph node cells from EAE rats that were cultured in the presence of MOGp33–55 (not shown).



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  		FIGURE 1. DNA vaccination-based anti-IL-27 Abs are highly specific. The Western blot shows that our DNA vaccination-based anti-IL-27 Ab binds mouse IL-27 p28 (lane 1; 27 kDa), but not recombinant mouse IL-18, IL-12, or TNF-{alpha} (lanes 2, 3, and 4, respectively). A, Coomassie Blue staining verifies the appearance of each cytokine on the loaded gel. B, Western blot showing that of these cytokines, our DNA vaccination-based anti-IL-27 Ab binds only mouse IL-27 p28. These Ab also bound natural mouse IL-27 (verified by sequencing) from supernatant of activated MOGp35–55-specific cultured primary draining lymph node cells (not shown).

 
We then determined the competence of this Ab to affect the dynamics of an ongoing disease. Starting on day 17, at the onset of disease, mice were subjected to subsequent administration of anti-IL-27 Abs, IgG from preimmunized rats, or PBS. Although both control groups (IgG and PBS) continued to develop severe EAE (Fig. 2A; mean maximal score of six mice ± SE, 3.3 ± 0.6 and 3.1 ± 0.3, respectively), in those treated with purified anti-p28 Ab, the development and progression of disease were markedly attenuated (mean maximal score, 0.7 ± 0.23; p < 0.001 compared with each control group). Histological analysis (Fig. 3 and Table I) of the lumbar spinal cord of these mice (day 30) clearly showed that disease suppression was accompanied by a significant reduction in parenchymal mononuclear cell infiltration (Table I; mean histological score, 0.8 ± 0.3 compared with 2.8 ± 0.3 and 2.8 ± 0.3 in control groups). In a subsequent experiment (Fig. 2B) we compared the abilities of anti-IL-27 Abs to suppress ongoing EAE to DNA vaccination-based Abs against IL-1{beta} and IL-18 produced by the same protocol. Each of these Abs could significantly suppress adjuvant-induced arthritis (not shown). Our results (Fig. 2B) clearly show that neutralizing IL-18 or IL-27 rapidly suppresses EAE in a similar manner. In contrast, neutralizing IL-1{beta} displays a much-moderated effect on the severity of disease that significantly differs (p < 0.001) from that obtained by neutralizing IL-18 or IL-27 (Fig. 2B). To determine whether the effects of these Abs are direct or indirect effect (i.e., suppression of the responses induced by the CFA with which active disease is induced), we determined the abilities of these Abs to suppress ongoing transferred EAE. Fig. 2C shows that similar to active disease, these Abs rapidly and most effectively suppress transferred EAE. Another impermanent question referred to the abilities of these Abs to induce long term resistance against EAE. We therefore conducted an experiment in which C57BL6 mice suffering from active EAE were treated with either a single dose or repeated administrations of anti-IL-27 neutralizing Abs. Fig. 4 shows that the beneficial effect of anti-IL-27 therapy in mice subjected to a single Ab administration was temporary and lasted for ~7–10 days.



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  		FIGURE 2. Anti-p28 Abs suppresses ongoing severe EAE. A, Four groups of 10 mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 µg/mouse of anti-p28 Ab ({blacksquare}), IgG obtained from naive Lewis rats ({blacktriangleup}), or PBS ({circ}). An observer blind to the experimental procedure scored EAE daily. The experiment summarized in Fig. 2 shows the results of one of three experiments performed under similar experimental conditions, with similar results. The mean maximal score ± SE represents six mice per group. The other four mice were killed on day 30 and subjected to histological evaluation (see Fig. 3). B, Five groups of six mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 µg of anti-p28 Ab/mouse ({blacksquare}), anti-IL-18 Ab (•), anti-IL-1{beta} Ab ({square}), IgG obtained from Lewis rats previously subjected to an empty plasmid administration ({blacktriangleup}), or PBS ({circ}). An observer blind to the experimental procedure scored EAE daily. Results are shown as the mean maximal score ± SE of six mice per group. C, Three groups of six mice each were subjected to induction of transferred EAE. Beginning at the onset of disease (day 5), these mice were repeatedly (every other day) administered 100 µg of anti-p28 Ab/mouse ({blacksquare}), IgG obtained from naive Lewis rats ({blacktriangleup}), or PBS ({circ}). An observer blind to the experimental procedure scored EAE daily. Results are shown as mean maximal score ± SE of six mice per group.

 


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  		FIGURE 3. Anti-IL-27 therapy reduces the histological score of EAE. Histological evaluation was conducted 30 days after disease induction. Lumbar spinal cord samples from naive mice or from EAE mice treated with PBS, IgG from naive mice, or anti-IL-27 p28 Abs were subjected to histological analysis (nine sections each group). The arrowheads point to the parenchymal mononuclear cell infiltration. The scale for mononuclear cell infiltration used was: 0, no mononuclear cell infiltration; 1, one to five perivascular lesions per section with minimal parenchymal infiltration; 2, five to 10 perivascular lesions per section with parenchymal infiltration; and 3, >10 perivascular lesions per section with extensive parenchymal infiltration. The mean histological score ± SE was calculated for each group.

 

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  		Table I. Histological score of the of the lumbar spinal cord of EAE mice treated with anti-IL-27 Abs

 


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  		FIGURE 4. The beneficial effect of anti-IL-27 is dependent on the continuing administration of protective Abs. Three groups of six mice each were subjected to active induction of EAE. Beginning 1 day after the onset of disease (day 17), these mice were treated with either a single dose of anti-p28 Ab (100 µg/mouse; {circ}) or with repeated administration (every other day) of this Ab ({blacktriangleup}) or PBS ({blacksquare}). An observer blind to the experimental procedure scored EAE daily. Results are shown as the mean maximal score ± SE of six mice per group.

 
Therapy with anti-IL-27 Abs decreases in vivo polarization of CD4+ T cells into Th1 and suppresses IFN-{gamma} production by Ag-specific T cells

To determine the possibility that the adoptive transfer of anti-IL-27 p28 Abs suppresses EAE by altering the in vivo polarization of CD4+ T cells. C57BL/6 mice were subjected to active induction of EAE and then to repeated administration (days 12, 14, and 16) of anti-IL-27 p28 Abs, PBS, or normal rat IgG. On day 17, cervical lymph node cells (that drain the autoimmune site) were obtained and subjected to FACS analysis (Fig. 5A) or were cocultured in the presence of 100 µM MOGp35–55. After 72 h of stimulation, supernatants were assayed for the protein levels of IFN-{gamma} (Fig. 5B) and IL-4 (not shown). Our results clearly show that repeated administration of anti-IL-27 Abs led to a significant reduction in Th1 polarization (Fig. 5A; ~80% of CD4+ Th1 cells in both control groups compared with 47.5% in anti-IL-27 p28-treated mice) and to a significant increase in IL-4lowIFN-{gamma}low-producing nonpolarized CD4+ T cells (~16% in control groups and 41.6% in treated mice). Analysis of cytokine production after stimulation in the presence of 100 µM MOGp35–55 showed a marked reduction in IFN-{gamma} production (Fig. 5B; 710 ± 50 and 740 ± 60 pg/ml in control groups compared with 240 ± 30 pg/ml in anti-IL-27-treated mice). IL-4 levels were <10 pg/ml in all groups, and no significant change in levels of TNF-{alpha} was found (not shown). The reduction in IFN-{gamma} production was accompanied by a marked reduction in Ag-specific proliferative response (Fig. 5C; 5200 ± 320 and 6200 ± 580 cpm with backgrounds of 1700 and 1760 cpm in control groups compared with 2600 ± 370 cpm, with background of 1700 cpm in anti-IL-27-treated mice; p < 0.0001).



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  		FIGURE 5. Protective administration of anti-IL-27 Abs decreases in vivo polarization of CD4+ T cells into Th1 and suppresses IFN-{gamma} production by Ag-specific T cells. C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 12, 14, and 16) of anti-IL-27 p28 Abs (100 µg), PBS, or normal rat IgG. On day 17 cervical lymph node cells (that drain the autoimmune site) were subjected to intracellular staining of IL-4 and IFN-{gamma}. A, FACS analysis of CD4+ T cells in this experiment. This experiment represents results obtained in three different independent experiments with very similar data. Subsequently, cervical lymph node T cells from these mice were cultured in the presence of 100 µM MOGp35–55. After 72 h of stimulation, supernatants were assayed for the protein level of IFN-{gamma} (B) and IL-4 (not shown). This experiment represents results obtained in three different independent experiments with very similar data.

 
It should be emphasized that intracellular staining of IFN-{gamma} was conducted on freshly isolated cervical lymph node T cells, whereas IFN-{gamma} levels were determined by ELISA after 3 days of activation with the target MOG Ag. It is plausible that after 3 days of in vitro priming, the relative number of Ag-specific T cells proliferating in response to their target Ag will dramatically increase; if they do produce IFN-{gamma}, its level would be extremely high, and if they do not, the level of this cytokine would be relatively low. Finally, the dramatic effect of anti-IL-27 Abs on in vitro production of IFN-{gamma} suggests that this cytokine promotes IFN-{gamma} production not only during the initial polarization of naive T cells in response to the target Ag (16), but also of already primed T cells to increase their Ag-specific proliferative response and IFN-{gamma} production. The paragraph below explores this possibility.

IL-27 acts on activated/memory CD4+ Th1 cells to increase their IFN-{gamma}, but not TNF-{alpha}, production

To determine whether IL-27 also acts on long term activated/memory T cells, we studied the effect of IL-27 on the proliferative response and cytokine production of our long term MOGp33–35-specific memory/effector (Th1) CD4+ T cell line. This line was cultured in the presence of rIL-27 p28 or recombinant {beta}-actin, which were constructed under the same conditions. The addition of rIL-27 p28 could very effectively increase IFN-{gamma} production by these cells (1580 ± 120 compared with 610 ± 70 pg/ml; p < 0.001). Our anti-IL-27 neutralizing Abs could successfully reverse this effect (Table I). Control IgG had no effect on this response (not shown). Thus, IL-27 can act as a potent proinflammatory mediator not only on naive cells undergoing pro-Th1 polarization (16), but also on T cells that have previously been polarized into Th1. The addition of IL-27 also significantly increased their proliferative response (Table II; ~70% increase; p < 0.001), but had no effect on TNF-{alpha} production by these cells (Table II). As expected, IL-4 was produced at very low levels by these Th1 cells. The addition of IL-27 did not increase the production of this cytokine by these cells. Taken together, these results show for the first time that the function of IL-27 is not limited to naive T cells.


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  		Table II. IL-27 elicits proliferation and IFN-{gamma} production by MOGp33–55-specific CD4+ T cell linea

 
To further determine whether IL-27 potentates the production of IFN-{gamma} in T cells, including long-term memory T cells we have conducted 2 sets of complementary experiments in which the function of IL-27 was neutralized in vivo (Fig. 6A) or in long term cultured T cells (Fig. 6B), and the relative number of IFN-{gamma}-producing T cells was determined by spot ELISA. In these experiments the size of each spot, representing the amount of cytokine produced by these cells, was also determined. We show in this study that blocking the in vivo function of IL-27 not only reduces the relative number of IFN-{gamma}-producing cells (Fig. 6A; 25 ± 3 compared with 152 ± 18 and 144 ± 15 spots/107 cells; p < 0.001), but also the amount (i.e., size of the spot) of cytokine produced by each cell (see Fig. 6A). Likewise, the addition of neutralizing Abs to IL-27 to long term cultured CD4+ T cells responding to their target Ag (MOG) significantly decreased the spot size of IFN-{gamma}-producing T cells (Fig. 6B; ~10-fold lower). This further substantiates the effect of IL-27 on the function of T cells, including those that have already been primed, and can explain in part the why neutralizing IL-27 rapidly and effectively suppresses an ongoing inflammatory autoimmune disease (Figs. 2 and 4).



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  		FIGURE 6. Neutralizing the function of IL-27 reduces IFN-{gamma} production by IFN-{gamma}-producing T cells. A, C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 3 and 6) of 100 µg of anti-IL-27 p28 Abs (group 3), PBS (group 2), or normal rat IgG (group 1). On day 9, spleen cells were subjected to spot ELISA as previously described (38 ). A, Relative number of positive spots per 107 cultured cells. The average size of positive spots was analyzed. B, The MOGp33–55-specific CD4+ T cell line was cultured with or without 100 µM MOGp33–55. Cultured cells were supplemented with anti-IL-27 Abs at a final concentration of 10 µg/ml ({square}), normal rat IgG ({blacksquare}), or PBS ({circ}). After 60 h of incubation, cells were plates in spot ELISA plates for an additional 24 h for the detection of IFN-{gamma}-positive spots (38 ). Number of positive spots (y-axis) and spot sizes (x-axis; logarithmic scale) determined as previously described (46 ).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current study shows that anti-IL-27-based Ab therapy can be used effectively to suppress an ongoing autoimmune disease (Figs. 2–4) and that the mechanism for this suppression includes an arrest of Th1 polarization (Fig. 5A) resulting in a significant reduction in the proliferative response and IFN-{gamma} production by Ag-specific T cells. We also show that IL-27 affects not only naive T cells undergoing Ag-specific activation, but also effector/memory Th1 cells (Table II). Finally, we show that the effect is dependent on repeated administration of these Abs (Fig. 4).

IL-27 is a newly defined family member of the long chain, four-helix bundle cytokines (16). This cytokine is an early product of activated APCs and drives rapid clonal expansion of naive CD4+ T cells. IL-27 synergizes with IL-12 to trigger IFN-{gamma} production of naive CD4+ T cells and mediates its biologic effects through the orphan cytokine receptor WSX-1/TCCR (16, 17, 18, 19, 20). This may suggest IL-27 as a potential relevant target for therapy of inflammatory autoimmune disease, particularly those in which Th1 cells are the key mediators of the inflammatory autoimmune process. In contrast, the finding that IL-27 acts primarily on naive, and not activated, T cells (16, 21, 22) may question the efficiency of anti-IL-27 therapy after the onset of disease. Additionally, a very recent study conducted in mice lacking the IL-27 p28 receptor (WSX-1–/– mice) shows that the absence of this particular receptor does not reduce the competence of these mice to provoke an inflammatory response against Toxoplasma gondii (39). Our results clearly show that neutralizing the p28 subunit of IL-27 exerts an anti-inflammatory effect that leads to a rapid suppression of an inflammatory autoimmune disease (Figs. 1 and 2). We clearly show that IL-27 also acts on primed T cells (Fig. 4, B and C), including long term memory/effector Th1 cells (Table I), to promote Ag-specific proliferation and IFN-{gamma} production. This could explain in part its key role in the regulation of autoimmune diseases and may also suggest a novel way for treating MS and possibly other T cell-mediated autoimmune diseases. To date, successful anti-inflammatory Ab therapy in EAE was usually dependent on repeated administration of the therapeutic Ab. For example, 12 years ago we defined the {alpha}4{beta}1 integrin as a major target for Ab therapy in EAE (40). These experiments have been very successfully extended to patients suffering from MS (41). In both, the beneficial effect is dependent on repeated administration of the protective Ab. It is plausible that the dynamics of these diseases is dependent on continuing polarization and activation of autoimmune cells replacing those that undergo apoptosis at the autoimmune site (30, 42). This may explain in part why a single administration of an Ab that redirects the function of autoimmune T cells or blocks their migratory properties cannot provide long-lasting resistance (Fig. 4).

Finally, alteration of the Th1/Th2 balance to Th2 either by interfering in costimulatory interactions (43) or by neutralizing IL-12, IL-18, or IFN-{gamma}-inducible protein-10 (CXCL10) (9, 10, 11) suppressed EAE. It is not clear what contributes more to this beneficial effect, the increased relative number of Ag-specific Th2 cells that produce IL-4 and IL-13 or the reduced relative number of Ag-specific inflammatory Th1 cells. Various studies, including ours, have previously shown that direct administration of Ag-specific Th2 cells does not transfer the beneficial effect obtained by altering the Ag-specific T cell polarization into Th2 (10, 44). This suggests that suppression of EAE by interfering in the Th1/Th2 balance suppresses the disease due to the reduction of the relative number of Ag-specific inflammatory T cells. The current study showing that anti-IL-27 therapy does not significantly select Th2 cells, but, rather, reduces the relative number of Ag-specific inflammatory T cells further suggests that suppression of Th1 function would be sufficient for suppressing EAE. We clearly show (Fig. 5) that neutralizing IL-27 leads to a significant reduction not only in the number of IFN-{gamma}-producing cells, but also in the amount of the cytokine produced by these cells. It has yet to be explored, however, whether the proinflammatory function of IL-27 is due to the direct amplification of IFN-{gamma} production by autoimmune T cells or is associated with other effects, as yet to be determined, of this cytokine on the function of these cells. Hence, from a clinically oriented perspective, such a therapy could be favorable in shifting the balance toward Th2, which might, over the long term, lead to the development of allergic responses (45).


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Israel Science Foundation, Chutic Fund for Brain Trauma Research, Israel Ministry of Health Chief Scientist, and the Technion Fund for Research Promotion. Back

2 Address correspondence and reprint requests to Dr. Nathan Karin, Department of Immunology. Bruce Rappaport Faculty of Medicine, Technion, P.O.B. 9697, Haifa 31096, Israel. E-mail address: nkarin{at}tx.technion.ac.il Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis. Back

Received for publication May 14, 2004. Accepted for publication September 15, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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