Differential Effects of IL-21 during Initiation and Progression of Autoimmunity against Neuroantigen
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免疫学杂志 2005年第5期
Abstract
The cytokine IL-21 is closely related to IL-2 and IL-15, a cytokine family that uses the common -chain for signaling. IL-21 is expressed by activated CD4+ T cells. We examined the role of IL-21 in the autoimmune disease experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. IL-21 administration before induction of EAE with a neuroantigen, myelin oligodendrocyte glycoprotein peptide 35-55, and adjuvant enhanced the inflammatory influx into the CNS, as well as the severity of EAE. Autoreactive T cells purified from IL-21-treated mice transferred more severe EAE than did the control encephalitogenic T cells. No such effects were observed when IL-21 was administered after EAE progressed. Additional studies demonstrated that IL-21 given before the induction of EAE boosted NK cell function, including secretion of IFN-. Depletion of NK cells abrogated the effect of IL-21. Therefore, IL-21, by affecting NK cells, has differential effects during the initiation and progression of autoimmune responses against neuroantigens.
Introduction
Interleukin-21 belongs to a cytokine family that uses the common -chain for signaling and is related structurally to cytokines IL-2, IL-4, and IL-15 (1). The IL-21R shares homology with the -chain of IL-2/IL-15Rs and forms a complex with the common -chain (2, 3). IL-21R is expressed on lymphoid tissues such as the thymus and lymph nodes, as well as on their components’ leukocytes and NK cells. In addition, IL-21R is found in bone marrow cells (2, 3). Yet, in contrast to the broad distribution of its receptor, IL-21 is produced only by CD4+ cells but not CD8+ T cells (1). On its own, IL-21 does not play an obvious mandatory role in lymphocyte development or function because IL-21R-deficient mice have a normal complement of developing mature T and B cells and lack severe immunopathology (reviewed in Ref.2). However, emerging evidence suggests that the IL-21R/ligand pair could have a broad and diverse responsibility in regulation of lymphoid cell function (2). For T cells, IL-21 appears to costimulate proliferation of anti-CD3-activated T cells, promote differentiation of VV 2 T cells into central memory CD45RO T cells, augment Th2 responses, and regulate T cell turnover (4, 5, 6, 7). IL-21 also regulates dendritic cells’ function by inhibiting their activation and maturation (8). IL-21 has significant influence on B cell function and cooperates with IL-4 for isotype switching (9, 10, 11). Using different activation signals, IL-21 can either induce B cell costimulation, arrest growth, or promote apoptosis (11). Within the NK cell lineage, IL-21 enhances maturation of human multipotent bone marrow progenitors and activates peripheral NK cells in the absence of other stimuli (1, 12). IL-21, in synergy with IL-15 or IL-18, enhances the functional maturation of human and murine NK cells (13, 14). In contrast, IL-21 seems to have an inhibitory effect on the IL-15-promoted expansion of murine NK cells but has no additional functions in the absence of activating signals (15).
The reported effects of IL-21, based primarily on in vitro studies, appear to be very broad and sometimes paradoxical. This led us to an important question: does IL-21 have differential effects during the initiation and progression of an immune response? We sought to address this issue in experimental autoimmune encephalomyelitis (EAE),3 an animal model of human multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG) is a myelin component of the CNS and is one of the candidate autoantigens that cause MS. MOG peptide 35-55/CFA immunization in C57BL/6 mice can activate the innate immune system and induce MOG-reactive Th1 cells (16, 17). These Th1 cells and other inflammatory cells and mediators can penetrate into the CNS, thereby producing inflammation and CNS syndrome. Induction and progression of EAE is a highly regulated events. Using this model, we demonstrated that IL-21 administration before disease induction enhanced inflammatory influx into the CNS and increased the severity of EAE. These effects were not observed when IL-21 was administered after EAE induction. In additional studies, the depletion of NK cells abrogated the effect of IL-21. Therefore, IL-21, at least in part by affecting NK cells, may have differential effects during initiation and progression of autoimmune responses against neuroantigens.
Materials and Methods
Mice
C57BL/6 (B6) mice, B6 B cell-deficient mice, μMt mice generated by targeted disruption of the μ H chain transmembrane exon (18) were purchased from The Jackson Laboratory. CD1D1-deficient mice (19), which lack a subset of NKT cells, were provided by Dr. L. Van Kaer (Vanderbilt University, Nashville, TN) and were backcrossed to B6 background for 11 generations. All mice were housed in the animal facilities of the Barrow Neurological Institute or The Scripps Rodent Colony. These mice were females and 8- to 10-wk of age at the experiment’s initiation.
Ags, Abs, and recombinant cytokines
The murine MOG35-55 peptide (M-E-V-G-W-Y-R-S-P-F-S-R-V-V-H-L-Y-R-N-G-K; Ref.16) was from Biosynth International. PK136 clone (anti-NK1.1; Ref.20) was obtained from American Type Culture Collection. Mouse IgG2a (Sigma-Aldrich) was used as an isotype control Ab for anti-NK1.1 Ab. For depletion of NK1.1+ cells in vivo, 100 μg of anti-NK1.1 mAb were injected i.p. into each mouse at day –2 postimmunization. Every 5–7 days thereafter, 50 μg of anti-NK1.1 mAb were injected i.p. until the termination of experiments (16, 21). Depletion was confirmed by flow cytometry with PE-NK1.1 Ab (BD Pharmingen). Mouse rIL-15 was purchased from BD Biosciences; murine rIL-21 was purchased from PeproTech. Each mouse received 0.01, 0.1, or 1 μg of IL-21 i.p. daily for 7 consecutive days.
Induction of EAE
EAE was induced in mice by s.c. injections into the flank and tail base of 200 μg of MOG peptide in CFA (Difco) containing 500 μg of heat-inactivated Mycobacterium tuberculosis on day 0. Supplemental injections of 200 ng of pertussis toxin were given i.v. on day 2 (List Biological Laboratories). The mice were observed daily for clinical signs of disease and scored on an arbitrary scale of 0–5 with graduations of 0.5 for intermediate scores (22): 0, no clinical signs; 1, flaccid tail; 2, hind limb weakness or abnormal gait; 3, complete hind limb paralysis; 4, complete hind limb paralysis with forelimb weakness or paralysis; and 5, moribund or deceased. For passively transferred EAE, mice were immunized with MOG35-55 as above. Fourteen days later, spleen cells suspensions from the immunized mice were cultured for 4 days with 10 μg/ml MOG35-55 and 15 U/ml IL-2 (PeproTech). After a wash with HBSS, 1 x 107 cells were injected i.v. into recipient mice.
Culture medium
Cells were suspended in DMEM (Invitrogen Life Technologies) supplemented with 1% (v/v) MEM (Invitrogen Life Technologies), 2 mM glutamine (Flow Laboratories), 50 IU/ml penicillin, and 50 mg/ml streptomycin and 10% (v/v) FCS (both from Invitrogen Life Technologies).
Cell isolation, sorting, and IFN- ELISPOT
Mononuclear cells (MNC) were obtained by mincing the spleens from test mice through a wire mesh. Splenic DX5+CD3– cells were sorted using a FACStarplus (BD Biosciences). DX5+CD3– spleen cells were >95% pure upon reanalysis by flow cytometry. A total of 2 x 105 NK cells was cultured in each well of a 96-well plate in the presence or absence of LPS (2 ng/ml), rIL-15 (10 ng/ml), and rIL-21 (10 ng/ml). A solid-phase ELISPOT assay was used to detect cytokine secretion at the single-cell level. Plastic plates (Dynatech Laboratories) were coated with 100 μl of IFN- to capture Ab (Innogenetics) at 15 μg/ml. After 48 h of culture, secreted and bound IFN- was visualized by application of biotinylated detector Ab (Innogenetics) and avidin-biotin complex (Dakopatts).
T cell proliferation
A total of 4 x 105 MNC was incubated in 200 μl of culture medium in 96-well round-bottom microtiter plates (Nunc). Ten-microliter aliquots of the MOG35-55 peptide or Con A (Sigma-Aldrich) were added into wells at final concentrations of 5, 10, 15, and 20 μg/ml (MOG35-55) or 5 μg/ml (Con A). After 4 days of incubation, the cells were pulsed for 18 h with 10-μl aliquots containing 1 μCi of [3H]methylthymidine (sp. act. 42 Ci/mmol; Amersham Biosciences). Cells were harvested onto glass fiber filters, and thymidine incorporation was measured.
Cytokine induction
Single-cell suspensions of MOG-primed spleen cells were cultured in the presence or absence of MOG35-55 (10 μg/ml), myelin basic protein (10 μg/ml), or Con A (5 μg/ml). NK cell stimulation and culture proceeded as described above. The supernatants were collected after 48 h in culture. IFN- and IL-4 production in culture supernatants were measured by optEIA kits (BD Pharmingen). The sensitivity of these ELISA was 31.3 pg/ml for IFN- and 7.8 pg/ml for IL-4.
Anti-MOG35-55 IgG Abs
Microtiter plates (Nunc) were coated with 100 μl/well of MOG35-55 peptide at a concentration of 5 μg/ml. MOG35-55-specific IgG and IgG isotypes were detected by ELISA using rabbit anti-mouse IgG1, IgG2a, or IgG2b (Dakopatts) as described previously (17).
Statistical analyses
Differences between groups were evaluated by ANOVA. Disease incidence and severity were analyzed by Fisher’s exact test and the Mann-Whitney U test, respectively.
Results
The timing of IL-21 administration determines its effect on MOG35-55-induced EAE
A single s.c. immunization of B6 mice with MOG peptide together with CFA and pertussis toxin induced moderate to severe EAE (mean maximum clinical score, 2.2 ± 0.6) in the majority of animals (84.2%, 16 of 19). The average time of disease onset was 18 days. Thereafter, the disease was associated with rapidly progressing, ascending paralysis appearing around day 20–25, which resulted in a protracted disease course. Remission began 25 days after primary immunization (Fig. 1A).
FIGURE 1. The timing of IL-21 administration determines the effects on MOG35-55 peptide-induced EAE. A, Mice were primed with MOG35-55/CFA and monitored for the development of EAE as described in Materials and Methods. IL-21 (0.1 μg/mouse) was injected s.c. for 7 consecutive days (*, n = 19). Mice were immunized with the MOG35-55 peptide during the last IL-21 injection. The same dosage and frequency of IL-21 were used for injections into another group of mice (?, n = 16) at day 7 after immunization. The mean clinical score of each group is plotted against the time after immunization. B, EAE in mice receiving spleen cells from donor mice treated with IL-21 before (*, n = 8) or after (?, n = 8) immunization with MOG35-55/CFA.
To examine whether IL-21 can affect the development of EAE and to determine an appropriate dose of recombinant murine IL-21, we first injected groups of B6 mice with three doses (0.01, 0.1, and 1 μg/mouse per injection). These injections were repeated for 7 consecutive days, and immunization with the MOG35-55 peptide began during the last IL-21 injection. Strikingly, 100% of the mice that received 0.1 or 1 μg/mouse before immunization with MOG35-55 developed EAE (14 of 14 and 8 of 8), with mean maximum clinical scores of 3.5 ± 0.4 and 3.4 ± 0.7, respectively (p < 0.05 for both comparisons with control mice). The disease of mice given 0.1 or 1 μg was significantly more severe than that of control mice (2.2 ± 0.6, 84.2%, 16 of 19) and of mice given 0.01 μg of IL-21 (2.4 ± 0.6, disease incidence 87.5%, 14 of 16; Fig. 1A). Additionally, the median days of EAE onset in mice treated with 0.1 and 1.0 μg of IL-21 were 9 and 12, respectively. Histological analysis of the inflammatory infiltrates in the IL-21-treated mice revealed that cellular infiltrates were more extensive than in control mice (Table I). Therefore, IL-21 given before EAE induction significantly enhanced EAE severity, as well as the related CNS inflammation. A dose of 0.1 μg/injection was chosen for subsequent study because its clinical effect was comparable to that with 1.0 μg, but 0.01 μg had no overt effect.
Table I. Survey of histological findings in cross-sections of spinal cord in EAE mice treated with IL-21
The capacity of IL-21 to modulate EAE during its initiation phase led us to investigate whether this cytokine had a similar effect after an autoimmune response to MOG became progressive. To this end, IL-21 (0.1 μg/injection) treatment was started on mice at day 9 postimmunization. This time point was chosen because, in our hands, vigorous MOG-reactive T cells have been induced, yet no EAE symptoms appeared. Surprisingly, as illustrated in Fig. 1, the incidence and severity of EAE were not significantly different between the control mice and IL-21-treated mice. The median day of EAE onset was 16 in treated animals vs 19 in controls. IL-21 had far less effect during EAE progression than at its initiation. Thus, the timing of IL-21 administration determined its effect on MOG35-55-induced EAE.
IL-21 alters the encephalitogenic potential of T cells
EAE is believed to be a Th1 cell-mediated disease of the CNS. MOG35-55-specific T cells are capable of transferring disease to recipients (23). We sought to address whether IL-21 can alter the encephalitogenic potential of autoreactive T cells by performing adoptive transfer experiments. Previously, 5 x 107 splenocytes from MOG35-55-sensitized mice injected i.v. caused severe EAE in the recipients starting on day 8. The disease lasted for 10 days, then spontaneously remitted to a mild or moderate form (16). We titrated the number of splenocytes from mice with EAE and found that transfer of 1 x 107 of such cells transferred only mild to moderate EAE (Fig. 1B). However, transferring the same number of splenocytes from IL-21-treated mice before immunization with MOG induced severe EAE (maximum clinical score 2.8 ± 0.4 vs control 1.7 ± 0.3, p < 0.05). When recipients of 1 x 107 splenocytes from animals treated with IL-21 after disease induction were compared with controls, both had a similarly low level of EAE (Fig. 1B). Therefore, IL-21 received before MOG challenge enhanced the encephalitogenic potential of T cells.
Effects of IL-21 on MOG-reactive Th cells
We next assessed Ag-specific spleen cell proliferation and cytokine induction in MOG35-55-sensitized control mice and mice treated with IL-21 before or after EAE induction. Spleen cells from these three groups of mice proliferated at a comparable magnitude in response to the MOG35-55 peptide (Fig. 2A), indicating that the IL-21 administration did not alter Ag-specific T cell proliferation in this model. However, compared with control mice, IL-21-pretreated mice had higher levels of IFN- production in response to the MOG35-55 peptide (Fig. 2B) but no significant difference in IL-4 production. Treatment with IL-21 after EAE induction did not significantly alter either IFN- or IL-4 production compared with that in control mice. These results indicated that IL-21 can augment autoreactive Th1 cells, without concomitant inhibition of Th2 cells.
FIGURE 2. Effects of IL-21 on MOG35-55-reactive T cell responses. Groups of mice were treated with IL-21 before or after immunization with the MOG35-55 peptide in CFA (Materials and Methods). A, Mice were killed at day 25 after immunization, and MNC were isolated from their spleens. Proliferative responses to different concentrations of the MOG35-55 peptide were assessed in each group (n = 4). The mean stimulation index plus SEM is plotted against the concentration of the MOG35-55 peptide. One representative experiment of three is shown. Background proliferation was 1,306 ± 110 cpm, and Con A-induced proliferation was 15,420 ± 4,300 cpm. No significant difference was found between control and IL-21-treated mice. B, Culture supernatants used for the cell proliferation assay were collected for determination of MOG35-55 peptide-specific cytokine production of T cells by ELISA. Spontaneous cytokine release: IFN-, 37 ± 15 pg/ml; IL-4, undetectable; and IL-10, 21 ± 11 pg/ml. No difference was revealed in spontaneous cytokine release between control mice and IL-21-treated mice. All results are expressed as mean values ± SEM. A and B represent one of two independent experiments with similar results (n = 4). A statistical evaluation was performed between the different experimental groups and control groups, respectively. *, p < 0.05.
Effects of IL-21 on autoantibody responses
CNS demyelination is considered to be an outcome of coordinated immune attacks initiated from both encephalomyelitic T cells and pathogenic autoantibodies in MOG-induced EAE (24). We tested this assumption by applying IL-21 pretreatment and evaluating its effect on B cell and autoantibody production. Compared with control mice, mice injected with IL-21 before EAE induction had significantly higher levels of circulating IgG and IgG2b Abs (Fig. 3), yet IL-21 treatment after immunization with MOG marginally elevated only the levels of IgG1 and IgG2a. In mice, IgG1 and IgG2a production is driven by Th2 cytokines such as IL-4, whereas IgG2b synthesis reflects a response to IFN-. The alteration of IgG subtype in recipients of IL-21 before vs after immunization may reflect changes in Th cytokine profiles. Although our study did not differentiate whether the elevated autoantibody response to MOG in IL-21-pretreated mice was a direct effect on B cells, on Th cells, or both, that response presumably contributed to the enhancement of EAE.
FIGURE 3. Autoantibody response to MOG35-55 in IL-21-treated mice. Mice used in experiments of Fig. 1A were bled periodically, and anti-MOG35-55 IgG and IgG isotypes were measured by ELISA on day 60 after immunization. All results are expressed as mean values ± SEM. A statistical evaluation was performed between the different experimental groups and control groups, respectively. *, p < 0.05.
Distinct action of IL-21 in NK cells during initiation and progression of EAE
The innate immune system, particularly NK cells, is thought to stimulate or suppress autoimmunity by direct interaction with APC or T cells or by release of immunoregulatory cytokines (25, 26). Of the latter, IL-21 enhances maturation from human bone marrow progenitors and activates peripheral NK cells in the absence of other stimuli (1). Paradoxically, IL-21 also has inhibitory effects on the IL-15-promoted expansion of mouse NK cells but requires activating signals for its functions (15). On the basis of these observations, we reasoned that the differential effects of IL-21 on the initiation vs progression of EAE could arise from an impact on NK cells. To do so, we first evaluated the prevalence of NK cells after IL-21 administration. Compared with control EAE mice, IL-21 before or after EAE induction did not have significantly altered numbers or percentages of NK cells (data not shown). Next, we examined the effect of IL-21 on the release of IFN- by NK cells because the timing of the release of this cytokine is critical for Th1 cell development (16). NK cells were sorted by FACS, after which IFN- production, either spontaneous or released by cultured NK cells in response to several stimuli, was quantified by ELISA. Compared with control mice, mice treated with IL-21 preceding immunization had a 2-fold increase in spontaneous IFN- production by NK cells (NK1.1+CD3–) and a 3-fold increase in IL-15 release after NK cells were stimulated with poly(I:C) (Fig. 4). However, the numbers of IFN--producing NK cells were comparable in controls and in mice treated with IL-21 after immunization. Therefore, IL-21 appears to act on NK cell functions (reflected by cytokine production) at different stages of autoimmune disease, rather than affecting the prevalence of NK cells at those times.
FIGURE 4. Effects of IL-21 on IFN- production by NK cells depend on the activation status of NK cells. NK cells were sorted by flow cytometry from spleens of control EAE mice or mice treated with IL-21 during or after immunization with MOG35-55 and CFA. NK cells were cultured for 48 h in the presence or absence of stimuli, and IFN- production by single NK cells in response to such stimuli was measured by ELISPOT. The figure represents one of two independent experiments (n = 4 mice/group). A statistical evaluation was performed between the different experimental groups and control groups, respectively. *, p < 0.05.
Removal of NK cells abrogates the effects of IL-21 on EAE
To assess whether the differing actions of IL-21 on NK cells at specific stages of the autoimmune response to MOG could account for differential effects of this cytokine in EAE, we performed a NK cell depletion study using mAb against NK1.1. As reported previously (16), a single injection of 150 μl of anti-NK1.1 eliminated 98% of NK1.1 cells; 50 μl of anti-NK1.1 given for 5 days maintained that depletion. When the NK1.1+ cell-depleted mice were then treated with IL-21 before MOG immunization, they developed EAE at a similar incidence and severity as control mice (Table II, group 3). Anti-NK1.1 abrogated the enhanced IFN- response to MOG35-55 (Table II, group 3). Depletion of NK1.1 cells 7 days after MOG immunization, just before IL-21 administration, did not produce a difference in either the severity of EAE or the MOG-specific Th1 cell (IFN- production) response (Table II, group 6). Therefore, the biological effects of IL-21 observed before EAE induction apparently depended on NK cells.
Table II. Removal of NK cells abrogates the effects of IL-21 on EAEa
Of note is that anti-NK1.1 Ab depletes NKT cells in addition to NK cells (16, 25). Although NKT cell deficiency does not significantly alter the course of MOG-induced EAE (F.-D. Shi, L. Van Kaer, and H.-G. Ljunggren, unpublished observations; Table II, group 10), NKT cells might mediate the effect of IL-21. To investigate this possibility, we treated CD1d1-deficient mice, which lack NKT cells (19), with IL-21 before or after immunization with MOG35-55. Table II illustrates that IL-21 treatment before, but not after, disease induction enhanced EAE severity (groups 11 and 12). Because the outcome of IL-21 treatment was similar to that in wild-type B6 mice, it is unlikely that NKT cells are responsible for the effects of IL-21 in this model.
B cells do not mediate the effects of IL-21 on MOG-induced EAE
A previous study demonstrated that the clinical course of EAE was similar in wild-type and B cell-deficient mice, except the latter mice seldom recovered (27), implying that B cells played a role in maintaining disease remission in this model. To evaluate whether B cells mediated the effects of IL-21 seen here, EAE presentation was examined in B cell-deficient mice (18) treated with IL-21. IL-21 exacerbated EAE when given before but not after immunization (Table II, groups 8 and 9). The magnitude of EAE in IL-21-treated, B cell-deficient mice was comparable with that of wild-type mice, arguing against the significant contribution of B cells in mediating the effects of IL-21.
IL-21 influences a broad range of lymphoid cells. In addition to NK cells, other cell types, e.g., APC, may also mediate the observed effects of IL-21 in MOG-induced EAE. Indeed, a recent study has reported that IL-21 can impact dendritic cell activation and maturation (7). However, our phenotyping of macrophages, dendritic cells, and B cells to determine the possible impact of IL-21 treatment indicated that expression levels of B-1, B7-2, CD40, and MHC class II were not significantly altered by IL-21 treatment in our model (data not shown).
Discussion
Emerging studies demonstrate that IL-21 has a broad role on diverse immune cells (2), there is little data concerning the role of for IL-21 in regulating (auto)immunity in vivo (28), and despite the limitations associated with cytokine injection, this approach allowed us to dissect the role of IL-21 at different stages of an organ-specific autoimmune disease, EAE. Our results indicate that IL-21, through affecting NK cells, alters the course of EAE during its initiation period but not during the subsequent progression of autoimmune responses against neuroantigens, hence, development of EAE.
A recent study by Wurster et al. (6) demonstrated that IL-21 is expressed preferentially by Th2 cells. Exposure of naive Th precursors to IL-21 inhibits IFN- production by preventing the precursors’ development into Th1 cells. Furthermore, IL-21R-deficient mice develop more robust delayed-type hypersensitivity reactions than their wild-type counterparts (6). These results suggest that IL-21 may relate more to Th2 cells yet can play a regulatory role for Th1 cells. In contrast, Strengell et al. (5) readily induced the expression of genes encoding IFN-, T-bet, IL-2R, IL-12R, IL-18R, and MyD88 on human T cells. These genes are important in activating Th1 responses. Although EAE is believed to be mediated by Th1 cells, the disease enhancement potential of IL-21 is not explained by the capacity of IL-21 to inhibit Th1 cells. Our results indicated that treating mice with IL-21 before disease induction actually enhanced the production of IFN- by T cells in response to MOG35-55. To the contrary, IL-21 treatment failed to augment the IFN- response during disease progression. These results suggest that the effects of IL-21 on autoreactive T cells occur within a discrete developmental window.
A question arises as to whether the differential effects of IL-21 during initiation vs progression of autoimmunity against MOG results from the direct action of IL-21 on autoreactive T cells or via other cell types that are influenced by IL-21. Our results cited here support the latter possibility because depletion of NK cells during disease induction abrogated the capacity of IL-21 to enhance CNS disease or to augment the MOG35-55-reactive Th1 response.
How would NK cells mediate the effects of IL-21 on MOG-induced EAE? To address this question, the role of NK cells in this particular model needs to be revisited. That innate immune responses influence the direction and magnitude of adaptive immune responses is a well-established concept. NK cells, by virtue of their ability to rapidly kill target cells and produce cytokines and chemokines, may be important in determining the genesis of autoimmunity (25, 26). However, available data concerning the contribution of NK cells in autoimmune disease are quite complex because in some circumstances, NK cells can either suppress or augment autoimmunity directly or indirectly (25, 26). A similar scenario has been reported in several EAE models. In MOG35-55-induced EAE of B6 mice, the depletion of NK cells by using anti-NK1.1 Ab derived from a PK136 clone enhanced the subsequent development of EAE (29). In contrast, the depletion of NK1.1 cells formerly decreased the incidence and severity of EAE (16). By using CD1D–/– mice, further study confirmed that the effects resulted from removal of NK cells but not NKT cells (16). Moreover, the transfer of NK cells to relatively NK cell-defective IL-18–/– mice partially restored their susceptibility to EAE, and transfer of IFN--deficient NK cells failed to restore that susceptibility. Thus, IFN- derived from NK cells is important for the development of MOG-reactive T cells and subsequently the emergence of CNS disease (16).
Until now, no clear clue has accounted for the discrepancy between these studies. A close survey of the experiment protocol used by Zhang et al. (29) revealed that they applied huge amounts of anti-NK1.1 Ab and that EAE induction began soon afterward. Because anti-NK1.1 can trigger a transient burst release of IFN- preceding the depletion of NK1.1 cells (30), one possibility is that a large concentration of IFN- present during naive T cell priming favors development of Th1 cells and thus promotes the onset of EAE (31, 32). If this possibility is true, the conclusion to be drawn from these two studies is that NK cell-derived IFN- is critical for genesis of MOG-reactive Th1 cells and hence development of EAE.
Our present study demonstrated that IL-21 treatment during immunization to induce EAE promoted IFN- production by NK cells, which might in turn favor Th1 commitment and worsen the EAE. This notion was supported additionally by our observation that removal of NK cells diminished the effects. NK cells are primed during induction of EAE, presumably by CFA and pertussis toxin (16). The fact that IL-21 failed to augment IFN- production after primary immunization leads to the prediction that the impact of IL-21 on NK cells depends largely on the activation status of these cells. In support, Kasaian et al. (15) demonstrated that IL-21 produced by CD4+ T cells actually resolved NK cell signals. However, the depletion of NK cells after disease induction does not significantly alter the course of EAE as it does during the T cell-priming period (16). Thus, NK cells may not be required for maintaining autoimmunity, either because of a feedback signal from T cells or lack of continuous NK stimuli.
Although the endogenous role of IL-21 in the autoimmune disease by using IL-21R–/– mice awaits upcoming studies, our results described here provide evidence that the effects of IL-21 in vivo are largely dependent on NK cells in MOG-induced EAE, on the status of these cells during the course of disease, and on the activating signals they receive. The observation that IL-21 has differential effects during the initiation and progression of autoimmunity additionally implies that this particular cytokine might be an important switch between innate immunity and autoimmunity.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Luc Van Kaer for providing the CD1D-deficient mice and Dianne DeNardo for assistance with animal experiments. We also thank the Barrow Neuroimmunology Team for their interest and sustained support for laboratory research programs and clinical trials.
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 grants from the Muscular Dystrophy Association (to F.-D.S.) and Barrow Neurological Foundation (to T.L.V., F.-D.S.).
2 Address correspondence and reprint requests to Dr. Fu-Dong Shi, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, 350 West Thomas Road, Phoenix, AZ 85013. E-mail address: fu-dong.shi{at}chw.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; MOG, myelin oligodendrocyte glycoprotein; MNC, mononuclear cell.
Received for publication September 17, 2004. Accepted for publication December 13, 2004.
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The cytokine IL-21 is closely related to IL-2 and IL-15, a cytokine family that uses the common -chain for signaling. IL-21 is expressed by activated CD4+ T cells. We examined the role of IL-21 in the autoimmune disease experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. IL-21 administration before induction of EAE with a neuroantigen, myelin oligodendrocyte glycoprotein peptide 35-55, and adjuvant enhanced the inflammatory influx into the CNS, as well as the severity of EAE. Autoreactive T cells purified from IL-21-treated mice transferred more severe EAE than did the control encephalitogenic T cells. No such effects were observed when IL-21 was administered after EAE progressed. Additional studies demonstrated that IL-21 given before the induction of EAE boosted NK cell function, including secretion of IFN-. Depletion of NK cells abrogated the effect of IL-21. Therefore, IL-21, by affecting NK cells, has differential effects during the initiation and progression of autoimmune responses against neuroantigens.
Introduction
Interleukin-21 belongs to a cytokine family that uses the common -chain for signaling and is related structurally to cytokines IL-2, IL-4, and IL-15 (1). The IL-21R shares homology with the -chain of IL-2/IL-15Rs and forms a complex with the common -chain (2, 3). IL-21R is expressed on lymphoid tissues such as the thymus and lymph nodes, as well as on their components’ leukocytes and NK cells. In addition, IL-21R is found in bone marrow cells (2, 3). Yet, in contrast to the broad distribution of its receptor, IL-21 is produced only by CD4+ cells but not CD8+ T cells (1). On its own, IL-21 does not play an obvious mandatory role in lymphocyte development or function because IL-21R-deficient mice have a normal complement of developing mature T and B cells and lack severe immunopathology (reviewed in Ref.2). However, emerging evidence suggests that the IL-21R/ligand pair could have a broad and diverse responsibility in regulation of lymphoid cell function (2). For T cells, IL-21 appears to costimulate proliferation of anti-CD3-activated T cells, promote differentiation of VV 2 T cells into central memory CD45RO T cells, augment Th2 responses, and regulate T cell turnover (4, 5, 6, 7). IL-21 also regulates dendritic cells’ function by inhibiting their activation and maturation (8). IL-21 has significant influence on B cell function and cooperates with IL-4 for isotype switching (9, 10, 11). Using different activation signals, IL-21 can either induce B cell costimulation, arrest growth, or promote apoptosis (11). Within the NK cell lineage, IL-21 enhances maturation of human multipotent bone marrow progenitors and activates peripheral NK cells in the absence of other stimuli (1, 12). IL-21, in synergy with IL-15 or IL-18, enhances the functional maturation of human and murine NK cells (13, 14). In contrast, IL-21 seems to have an inhibitory effect on the IL-15-promoted expansion of murine NK cells but has no additional functions in the absence of activating signals (15).
The reported effects of IL-21, based primarily on in vitro studies, appear to be very broad and sometimes paradoxical. This led us to an important question: does IL-21 have differential effects during the initiation and progression of an immune response? We sought to address this issue in experimental autoimmune encephalomyelitis (EAE),3 an animal model of human multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG) is a myelin component of the CNS and is one of the candidate autoantigens that cause MS. MOG peptide 35-55/CFA immunization in C57BL/6 mice can activate the innate immune system and induce MOG-reactive Th1 cells (16, 17). These Th1 cells and other inflammatory cells and mediators can penetrate into the CNS, thereby producing inflammation and CNS syndrome. Induction and progression of EAE is a highly regulated events. Using this model, we demonstrated that IL-21 administration before disease induction enhanced inflammatory influx into the CNS and increased the severity of EAE. These effects were not observed when IL-21 was administered after EAE induction. In additional studies, the depletion of NK cells abrogated the effect of IL-21. Therefore, IL-21, at least in part by affecting NK cells, may have differential effects during initiation and progression of autoimmune responses against neuroantigens.
Materials and Methods
Mice
C57BL/6 (B6) mice, B6 B cell-deficient mice, μMt mice generated by targeted disruption of the μ H chain transmembrane exon (18) were purchased from The Jackson Laboratory. CD1D1-deficient mice (19), which lack a subset of NKT cells, were provided by Dr. L. Van Kaer (Vanderbilt University, Nashville, TN) and were backcrossed to B6 background for 11 generations. All mice were housed in the animal facilities of the Barrow Neurological Institute or The Scripps Rodent Colony. These mice were females and 8- to 10-wk of age at the experiment’s initiation.
Ags, Abs, and recombinant cytokines
The murine MOG35-55 peptide (M-E-V-G-W-Y-R-S-P-F-S-R-V-V-H-L-Y-R-N-G-K; Ref.16) was from Biosynth International. PK136 clone (anti-NK1.1; Ref.20) was obtained from American Type Culture Collection. Mouse IgG2a (Sigma-Aldrich) was used as an isotype control Ab for anti-NK1.1 Ab. For depletion of NK1.1+ cells in vivo, 100 μg of anti-NK1.1 mAb were injected i.p. into each mouse at day –2 postimmunization. Every 5–7 days thereafter, 50 μg of anti-NK1.1 mAb were injected i.p. until the termination of experiments (16, 21). Depletion was confirmed by flow cytometry with PE-NK1.1 Ab (BD Pharmingen). Mouse rIL-15 was purchased from BD Biosciences; murine rIL-21 was purchased from PeproTech. Each mouse received 0.01, 0.1, or 1 μg of IL-21 i.p. daily for 7 consecutive days.
Induction of EAE
EAE was induced in mice by s.c. injections into the flank and tail base of 200 μg of MOG peptide in CFA (Difco) containing 500 μg of heat-inactivated Mycobacterium tuberculosis on day 0. Supplemental injections of 200 ng of pertussis toxin were given i.v. on day 2 (List Biological Laboratories). The mice were observed daily for clinical signs of disease and scored on an arbitrary scale of 0–5 with graduations of 0.5 for intermediate scores (22): 0, no clinical signs; 1, flaccid tail; 2, hind limb weakness or abnormal gait; 3, complete hind limb paralysis; 4, complete hind limb paralysis with forelimb weakness or paralysis; and 5, moribund or deceased. For passively transferred EAE, mice were immunized with MOG35-55 as above. Fourteen days later, spleen cells suspensions from the immunized mice were cultured for 4 days with 10 μg/ml MOG35-55 and 15 U/ml IL-2 (PeproTech). After a wash with HBSS, 1 x 107 cells were injected i.v. into recipient mice.
Culture medium
Cells were suspended in DMEM (Invitrogen Life Technologies) supplemented with 1% (v/v) MEM (Invitrogen Life Technologies), 2 mM glutamine (Flow Laboratories), 50 IU/ml penicillin, and 50 mg/ml streptomycin and 10% (v/v) FCS (both from Invitrogen Life Technologies).
Cell isolation, sorting, and IFN- ELISPOT
Mononuclear cells (MNC) were obtained by mincing the spleens from test mice through a wire mesh. Splenic DX5+CD3– cells were sorted using a FACStarplus (BD Biosciences). DX5+CD3– spleen cells were >95% pure upon reanalysis by flow cytometry. A total of 2 x 105 NK cells was cultured in each well of a 96-well plate in the presence or absence of LPS (2 ng/ml), rIL-15 (10 ng/ml), and rIL-21 (10 ng/ml). A solid-phase ELISPOT assay was used to detect cytokine secretion at the single-cell level. Plastic plates (Dynatech Laboratories) were coated with 100 μl of IFN- to capture Ab (Innogenetics) at 15 μg/ml. After 48 h of culture, secreted and bound IFN- was visualized by application of biotinylated detector Ab (Innogenetics) and avidin-biotin complex (Dakopatts).
T cell proliferation
A total of 4 x 105 MNC was incubated in 200 μl of culture medium in 96-well round-bottom microtiter plates (Nunc). Ten-microliter aliquots of the MOG35-55 peptide or Con A (Sigma-Aldrich) were added into wells at final concentrations of 5, 10, 15, and 20 μg/ml (MOG35-55) or 5 μg/ml (Con A). After 4 days of incubation, the cells were pulsed for 18 h with 10-μl aliquots containing 1 μCi of [3H]methylthymidine (sp. act. 42 Ci/mmol; Amersham Biosciences). Cells were harvested onto glass fiber filters, and thymidine incorporation was measured.
Cytokine induction
Single-cell suspensions of MOG-primed spleen cells were cultured in the presence or absence of MOG35-55 (10 μg/ml), myelin basic protein (10 μg/ml), or Con A (5 μg/ml). NK cell stimulation and culture proceeded as described above. The supernatants were collected after 48 h in culture. IFN- and IL-4 production in culture supernatants were measured by optEIA kits (BD Pharmingen). The sensitivity of these ELISA was 31.3 pg/ml for IFN- and 7.8 pg/ml for IL-4.
Anti-MOG35-55 IgG Abs
Microtiter plates (Nunc) were coated with 100 μl/well of MOG35-55 peptide at a concentration of 5 μg/ml. MOG35-55-specific IgG and IgG isotypes were detected by ELISA using rabbit anti-mouse IgG1, IgG2a, or IgG2b (Dakopatts) as described previously (17).
Statistical analyses
Differences between groups were evaluated by ANOVA. Disease incidence and severity were analyzed by Fisher’s exact test and the Mann-Whitney U test, respectively.
Results
The timing of IL-21 administration determines its effect on MOG35-55-induced EAE
A single s.c. immunization of B6 mice with MOG peptide together with CFA and pertussis toxin induced moderate to severe EAE (mean maximum clinical score, 2.2 ± 0.6) in the majority of animals (84.2%, 16 of 19). The average time of disease onset was 18 days. Thereafter, the disease was associated with rapidly progressing, ascending paralysis appearing around day 20–25, which resulted in a protracted disease course. Remission began 25 days after primary immunization (Fig. 1A).
FIGURE 1. The timing of IL-21 administration determines the effects on MOG35-55 peptide-induced EAE. A, Mice were primed with MOG35-55/CFA and monitored for the development of EAE as described in Materials and Methods. IL-21 (0.1 μg/mouse) was injected s.c. for 7 consecutive days (*, n = 19). Mice were immunized with the MOG35-55 peptide during the last IL-21 injection. The same dosage and frequency of IL-21 were used for injections into another group of mice (?, n = 16) at day 7 after immunization. The mean clinical score of each group is plotted against the time after immunization. B, EAE in mice receiving spleen cells from donor mice treated with IL-21 before (*, n = 8) or after (?, n = 8) immunization with MOG35-55/CFA.
To examine whether IL-21 can affect the development of EAE and to determine an appropriate dose of recombinant murine IL-21, we first injected groups of B6 mice with three doses (0.01, 0.1, and 1 μg/mouse per injection). These injections were repeated for 7 consecutive days, and immunization with the MOG35-55 peptide began during the last IL-21 injection. Strikingly, 100% of the mice that received 0.1 or 1 μg/mouse before immunization with MOG35-55 developed EAE (14 of 14 and 8 of 8), with mean maximum clinical scores of 3.5 ± 0.4 and 3.4 ± 0.7, respectively (p < 0.05 for both comparisons with control mice). The disease of mice given 0.1 or 1 μg was significantly more severe than that of control mice (2.2 ± 0.6, 84.2%, 16 of 19) and of mice given 0.01 μg of IL-21 (2.4 ± 0.6, disease incidence 87.5%, 14 of 16; Fig. 1A). Additionally, the median days of EAE onset in mice treated with 0.1 and 1.0 μg of IL-21 were 9 and 12, respectively. Histological analysis of the inflammatory infiltrates in the IL-21-treated mice revealed that cellular infiltrates were more extensive than in control mice (Table I). Therefore, IL-21 given before EAE induction significantly enhanced EAE severity, as well as the related CNS inflammation. A dose of 0.1 μg/injection was chosen for subsequent study because its clinical effect was comparable to that with 1.0 μg, but 0.01 μg had no overt effect.
Table I. Survey of histological findings in cross-sections of spinal cord in EAE mice treated with IL-21
The capacity of IL-21 to modulate EAE during its initiation phase led us to investigate whether this cytokine had a similar effect after an autoimmune response to MOG became progressive. To this end, IL-21 (0.1 μg/injection) treatment was started on mice at day 9 postimmunization. This time point was chosen because, in our hands, vigorous MOG-reactive T cells have been induced, yet no EAE symptoms appeared. Surprisingly, as illustrated in Fig. 1, the incidence and severity of EAE were not significantly different between the control mice and IL-21-treated mice. The median day of EAE onset was 16 in treated animals vs 19 in controls. IL-21 had far less effect during EAE progression than at its initiation. Thus, the timing of IL-21 administration determined its effect on MOG35-55-induced EAE.
IL-21 alters the encephalitogenic potential of T cells
EAE is believed to be a Th1 cell-mediated disease of the CNS. MOG35-55-specific T cells are capable of transferring disease to recipients (23). We sought to address whether IL-21 can alter the encephalitogenic potential of autoreactive T cells by performing adoptive transfer experiments. Previously, 5 x 107 splenocytes from MOG35-55-sensitized mice injected i.v. caused severe EAE in the recipients starting on day 8. The disease lasted for 10 days, then spontaneously remitted to a mild or moderate form (16). We titrated the number of splenocytes from mice with EAE and found that transfer of 1 x 107 of such cells transferred only mild to moderate EAE (Fig. 1B). However, transferring the same number of splenocytes from IL-21-treated mice before immunization with MOG induced severe EAE (maximum clinical score 2.8 ± 0.4 vs control 1.7 ± 0.3, p < 0.05). When recipients of 1 x 107 splenocytes from animals treated with IL-21 after disease induction were compared with controls, both had a similarly low level of EAE (Fig. 1B). Therefore, IL-21 received before MOG challenge enhanced the encephalitogenic potential of T cells.
Effects of IL-21 on MOG-reactive Th cells
We next assessed Ag-specific spleen cell proliferation and cytokine induction in MOG35-55-sensitized control mice and mice treated with IL-21 before or after EAE induction. Spleen cells from these three groups of mice proliferated at a comparable magnitude in response to the MOG35-55 peptide (Fig. 2A), indicating that the IL-21 administration did not alter Ag-specific T cell proliferation in this model. However, compared with control mice, IL-21-pretreated mice had higher levels of IFN- production in response to the MOG35-55 peptide (Fig. 2B) but no significant difference in IL-4 production. Treatment with IL-21 after EAE induction did not significantly alter either IFN- or IL-4 production compared with that in control mice. These results indicated that IL-21 can augment autoreactive Th1 cells, without concomitant inhibition of Th2 cells.
FIGURE 2. Effects of IL-21 on MOG35-55-reactive T cell responses. Groups of mice were treated with IL-21 before or after immunization with the MOG35-55 peptide in CFA (Materials and Methods). A, Mice were killed at day 25 after immunization, and MNC were isolated from their spleens. Proliferative responses to different concentrations of the MOG35-55 peptide were assessed in each group (n = 4). The mean stimulation index plus SEM is plotted against the concentration of the MOG35-55 peptide. One representative experiment of three is shown. Background proliferation was 1,306 ± 110 cpm, and Con A-induced proliferation was 15,420 ± 4,300 cpm. No significant difference was found between control and IL-21-treated mice. B, Culture supernatants used for the cell proliferation assay were collected for determination of MOG35-55 peptide-specific cytokine production of T cells by ELISA. Spontaneous cytokine release: IFN-, 37 ± 15 pg/ml; IL-4, undetectable; and IL-10, 21 ± 11 pg/ml. No difference was revealed in spontaneous cytokine release between control mice and IL-21-treated mice. All results are expressed as mean values ± SEM. A and B represent one of two independent experiments with similar results (n = 4). A statistical evaluation was performed between the different experimental groups and control groups, respectively. *, p < 0.05.
Effects of IL-21 on autoantibody responses
CNS demyelination is considered to be an outcome of coordinated immune attacks initiated from both encephalomyelitic T cells and pathogenic autoantibodies in MOG-induced EAE (24). We tested this assumption by applying IL-21 pretreatment and evaluating its effect on B cell and autoantibody production. Compared with control mice, mice injected with IL-21 before EAE induction had significantly higher levels of circulating IgG and IgG2b Abs (Fig. 3), yet IL-21 treatment after immunization with MOG marginally elevated only the levels of IgG1 and IgG2a. In mice, IgG1 and IgG2a production is driven by Th2 cytokines such as IL-4, whereas IgG2b synthesis reflects a response to IFN-. The alteration of IgG subtype in recipients of IL-21 before vs after immunization may reflect changes in Th cytokine profiles. Although our study did not differentiate whether the elevated autoantibody response to MOG in IL-21-pretreated mice was a direct effect on B cells, on Th cells, or both, that response presumably contributed to the enhancement of EAE.
FIGURE 3. Autoantibody response to MOG35-55 in IL-21-treated mice. Mice used in experiments of Fig. 1A were bled periodically, and anti-MOG35-55 IgG and IgG isotypes were measured by ELISA on day 60 after immunization. All results are expressed as mean values ± SEM. A statistical evaluation was performed between the different experimental groups and control groups, respectively. *, p < 0.05.
Distinct action of IL-21 in NK cells during initiation and progression of EAE
The innate immune system, particularly NK cells, is thought to stimulate or suppress autoimmunity by direct interaction with APC or T cells or by release of immunoregulatory cytokines (25, 26). Of the latter, IL-21 enhances maturation from human bone marrow progenitors and activates peripheral NK cells in the absence of other stimuli (1). Paradoxically, IL-21 also has inhibitory effects on the IL-15-promoted expansion of mouse NK cells but requires activating signals for its functions (15). On the basis of these observations, we reasoned that the differential effects of IL-21 on the initiation vs progression of EAE could arise from an impact on NK cells. To do so, we first evaluated the prevalence of NK cells after IL-21 administration. Compared with control EAE mice, IL-21 before or after EAE induction did not have significantly altered numbers or percentages of NK cells (data not shown). Next, we examined the effect of IL-21 on the release of IFN- by NK cells because the timing of the release of this cytokine is critical for Th1 cell development (16). NK cells were sorted by FACS, after which IFN- production, either spontaneous or released by cultured NK cells in response to several stimuli, was quantified by ELISA. Compared with control mice, mice treated with IL-21 preceding immunization had a 2-fold increase in spontaneous IFN- production by NK cells (NK1.1+CD3–) and a 3-fold increase in IL-15 release after NK cells were stimulated with poly(I:C) (Fig. 4). However, the numbers of IFN--producing NK cells were comparable in controls and in mice treated with IL-21 after immunization. Therefore, IL-21 appears to act on NK cell functions (reflected by cytokine production) at different stages of autoimmune disease, rather than affecting the prevalence of NK cells at those times.
FIGURE 4. Effects of IL-21 on IFN- production by NK cells depend on the activation status of NK cells. NK cells were sorted by flow cytometry from spleens of control EAE mice or mice treated with IL-21 during or after immunization with MOG35-55 and CFA. NK cells were cultured for 48 h in the presence or absence of stimuli, and IFN- production by single NK cells in response to such stimuli was measured by ELISPOT. The figure represents one of two independent experiments (n = 4 mice/group). A statistical evaluation was performed between the different experimental groups and control groups, respectively. *, p < 0.05.
Removal of NK cells abrogates the effects of IL-21 on EAE
To assess whether the differing actions of IL-21 on NK cells at specific stages of the autoimmune response to MOG could account for differential effects of this cytokine in EAE, we performed a NK cell depletion study using mAb against NK1.1. As reported previously (16), a single injection of 150 μl of anti-NK1.1 eliminated 98% of NK1.1 cells; 50 μl of anti-NK1.1 given for 5 days maintained that depletion. When the NK1.1+ cell-depleted mice were then treated with IL-21 before MOG immunization, they developed EAE at a similar incidence and severity as control mice (Table II, group 3). Anti-NK1.1 abrogated the enhanced IFN- response to MOG35-55 (Table II, group 3). Depletion of NK1.1 cells 7 days after MOG immunization, just before IL-21 administration, did not produce a difference in either the severity of EAE or the MOG-specific Th1 cell (IFN- production) response (Table II, group 6). Therefore, the biological effects of IL-21 observed before EAE induction apparently depended on NK cells.
Table II. Removal of NK cells abrogates the effects of IL-21 on EAEa
Of note is that anti-NK1.1 Ab depletes NKT cells in addition to NK cells (16, 25). Although NKT cell deficiency does not significantly alter the course of MOG-induced EAE (F.-D. Shi, L. Van Kaer, and H.-G. Ljunggren, unpublished observations; Table II, group 10), NKT cells might mediate the effect of IL-21. To investigate this possibility, we treated CD1d1-deficient mice, which lack NKT cells (19), with IL-21 before or after immunization with MOG35-55. Table II illustrates that IL-21 treatment before, but not after, disease induction enhanced EAE severity (groups 11 and 12). Because the outcome of IL-21 treatment was similar to that in wild-type B6 mice, it is unlikely that NKT cells are responsible for the effects of IL-21 in this model.
B cells do not mediate the effects of IL-21 on MOG-induced EAE
A previous study demonstrated that the clinical course of EAE was similar in wild-type and B cell-deficient mice, except the latter mice seldom recovered (27), implying that B cells played a role in maintaining disease remission in this model. To evaluate whether B cells mediated the effects of IL-21 seen here, EAE presentation was examined in B cell-deficient mice (18) treated with IL-21. IL-21 exacerbated EAE when given before but not after immunization (Table II, groups 8 and 9). The magnitude of EAE in IL-21-treated, B cell-deficient mice was comparable with that of wild-type mice, arguing against the significant contribution of B cells in mediating the effects of IL-21.
IL-21 influences a broad range of lymphoid cells. In addition to NK cells, other cell types, e.g., APC, may also mediate the observed effects of IL-21 in MOG-induced EAE. Indeed, a recent study has reported that IL-21 can impact dendritic cell activation and maturation (7). However, our phenotyping of macrophages, dendritic cells, and B cells to determine the possible impact of IL-21 treatment indicated that expression levels of B-1, B7-2, CD40, and MHC class II were not significantly altered by IL-21 treatment in our model (data not shown).
Discussion
Emerging studies demonstrate that IL-21 has a broad role on diverse immune cells (2), there is little data concerning the role of for IL-21 in regulating (auto)immunity in vivo (28), and despite the limitations associated with cytokine injection, this approach allowed us to dissect the role of IL-21 at different stages of an organ-specific autoimmune disease, EAE. Our results indicate that IL-21, through affecting NK cells, alters the course of EAE during its initiation period but not during the subsequent progression of autoimmune responses against neuroantigens, hence, development of EAE.
A recent study by Wurster et al. (6) demonstrated that IL-21 is expressed preferentially by Th2 cells. Exposure of naive Th precursors to IL-21 inhibits IFN- production by preventing the precursors’ development into Th1 cells. Furthermore, IL-21R-deficient mice develop more robust delayed-type hypersensitivity reactions than their wild-type counterparts (6). These results suggest that IL-21 may relate more to Th2 cells yet can play a regulatory role for Th1 cells. In contrast, Strengell et al. (5) readily induced the expression of genes encoding IFN-, T-bet, IL-2R, IL-12R, IL-18R, and MyD88 on human T cells. These genes are important in activating Th1 responses. Although EAE is believed to be mediated by Th1 cells, the disease enhancement potential of IL-21 is not explained by the capacity of IL-21 to inhibit Th1 cells. Our results indicated that treating mice with IL-21 before disease induction actually enhanced the production of IFN- by T cells in response to MOG35-55. To the contrary, IL-21 treatment failed to augment the IFN- response during disease progression. These results suggest that the effects of IL-21 on autoreactive T cells occur within a discrete developmental window.
A question arises as to whether the differential effects of IL-21 during initiation vs progression of autoimmunity against MOG results from the direct action of IL-21 on autoreactive T cells or via other cell types that are influenced by IL-21. Our results cited here support the latter possibility because depletion of NK cells during disease induction abrogated the capacity of IL-21 to enhance CNS disease or to augment the MOG35-55-reactive Th1 response.
How would NK cells mediate the effects of IL-21 on MOG-induced EAE? To address this question, the role of NK cells in this particular model needs to be revisited. That innate immune responses influence the direction and magnitude of adaptive immune responses is a well-established concept. NK cells, by virtue of their ability to rapidly kill target cells and produce cytokines and chemokines, may be important in determining the genesis of autoimmunity (25, 26). However, available data concerning the contribution of NK cells in autoimmune disease are quite complex because in some circumstances, NK cells can either suppress or augment autoimmunity directly or indirectly (25, 26). A similar scenario has been reported in several EAE models. In MOG35-55-induced EAE of B6 mice, the depletion of NK cells by using anti-NK1.1 Ab derived from a PK136 clone enhanced the subsequent development of EAE (29). In contrast, the depletion of NK1.1 cells formerly decreased the incidence and severity of EAE (16). By using CD1D–/– mice, further study confirmed that the effects resulted from removal of NK cells but not NKT cells (16). Moreover, the transfer of NK cells to relatively NK cell-defective IL-18–/– mice partially restored their susceptibility to EAE, and transfer of IFN--deficient NK cells failed to restore that susceptibility. Thus, IFN- derived from NK cells is important for the development of MOG-reactive T cells and subsequently the emergence of CNS disease (16).
Until now, no clear clue has accounted for the discrepancy between these studies. A close survey of the experiment protocol used by Zhang et al. (29) revealed that they applied huge amounts of anti-NK1.1 Ab and that EAE induction began soon afterward. Because anti-NK1.1 can trigger a transient burst release of IFN- preceding the depletion of NK1.1 cells (30), one possibility is that a large concentration of IFN- present during naive T cell priming favors development of Th1 cells and thus promotes the onset of EAE (31, 32). If this possibility is true, the conclusion to be drawn from these two studies is that NK cell-derived IFN- is critical for genesis of MOG-reactive Th1 cells and hence development of EAE.
Our present study demonstrated that IL-21 treatment during immunization to induce EAE promoted IFN- production by NK cells, which might in turn favor Th1 commitment and worsen the EAE. This notion was supported additionally by our observation that removal of NK cells diminished the effects. NK cells are primed during induction of EAE, presumably by CFA and pertussis toxin (16). The fact that IL-21 failed to augment IFN- production after primary immunization leads to the prediction that the impact of IL-21 on NK cells depends largely on the activation status of these cells. In support, Kasaian et al. (15) demonstrated that IL-21 produced by CD4+ T cells actually resolved NK cell signals. However, the depletion of NK cells after disease induction does not significantly alter the course of EAE as it does during the T cell-priming period (16). Thus, NK cells may not be required for maintaining autoimmunity, either because of a feedback signal from T cells or lack of continuous NK stimuli.
Although the endogenous role of IL-21 in the autoimmune disease by using IL-21R–/– mice awaits upcoming studies, our results described here provide evidence that the effects of IL-21 in vivo are largely dependent on NK cells in MOG-induced EAE, on the status of these cells during the course of disease, and on the activating signals they receive. The observation that IL-21 has differential effects during the initiation and progression of autoimmunity additionally implies that this particular cytokine might be an important switch between innate immunity and autoimmunity.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Luc Van Kaer for providing the CD1D-deficient mice and Dianne DeNardo for assistance with animal experiments. We also thank the Barrow Neuroimmunology Team for their interest and sustained support for laboratory research programs and clinical trials.
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 grants from the Muscular Dystrophy Association (to F.-D.S.) and Barrow Neurological Foundation (to T.L.V., F.-D.S.).
2 Address correspondence and reprint requests to Dr. Fu-Dong Shi, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, 350 West Thomas Road, Phoenix, AZ 85013. E-mail address: fu-dong.shi{at}chw.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; MOG, myelin oligodendrocyte glycoprotein; MNC, mononuclear cell.
Received for publication September 17, 2004. Accepted for publication December 13, 2004.
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