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Gene Deletion Leads to Augmented and Chronic Demyelinating Experimental Autoimmune Encephalomyelitis1
* Sections for Medical Inflammation Research and
Immunology, Department of Cell and Molecular Biology, University of Lund, Lund, Sweden
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Abstract |
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in multiple sclerosis (MS) patients are still obscure, here we have investigated the effects of IFN- gene disruption on the commonly used animal model for MS, experimental autoimmune encephalomyelitis (EAE). We show that IFN- knockout (KO) mice are more susceptible to EAE than their wild-type (wt) littermates; they develop more severe and chronic neurological symptoms with more extensive CNS inflammation and demyelination. However, there was no discrepancy observed between wt and KO mice regarding the capacity of T cells to proliferate or produce IFN-
in response to recall Ag. Consequently, we addressed the effect of IFN- on encephalitogenic T cell development and the disease initiation phase by passive transfer of autoreactive T cells from KO or wt littermates to both groups of mice. Interestingly, IFN- KO mice acquired a higher incidence and augmented EAE regardless of the source of T cells. This shows that the anti-inflammatory effect of endogenous IFN- is predominantly exerted on the effector phase of the disease. Histopathological investigations of CNS in the effector phase revealed an extensive microglia activation and TNF-
production in IFN- KO mice; this was virtually absent in wt littermates. This coincided with an increase in effector functions of T cells in IFN- KO mice, as measured by IFN- and IL-4 production. We suggest that lack of endogenous IFN- in CNS leads to augmented microglia activation, resulting in a sustained inflammation, cytokine production, and tissue damage with consequent chronic neurological deficits. |
Introduction |
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and IFN- together compose the two major kinds of type I IFNs. These are distinct from the type II IFN (IFN-) being produced by different cell types and sharing a distinct receptor, the IFN-/ receptor. IFN- is mainly produced by fibroblasts, macrophages, and dendritic cells in response to viral infection, dsRNA, or non-vertebrate unmethylated DNA containing CpG motifs (1, 2, 3). IFN- is a highly pleiotropic cytokine with many immunomodulatory effects on the cells of the innate as well as the adaptive immune system and because of this has been used as a therapy in several diseases, such as viral infections and cancer (4). One of the more recent diseases treated with IFN- is multiple sclerosis (MS).3
MS is considered an autoimmune disease characterized by inflammation and demyelination of the CNS leading to chronic neurologic disturbances (5, 6). One of the most commonly used therapies for MS at present is systemic administration of IFN-. IFN- has been shown to reduce the frequency of clinical exacerbations, the progression of disability, and cumulative disease burden as assessed by serial magnetic resonance imaging (7, 8). However, despite the fact that IFN- can clearly provide MS patients with clinical benefits, the effect is only partial and is not curative. Furthermore, there is a large group of patients who do not respond to the treatment. In addition, the mechanisms behind the action of IFN- remain largely unknown impeding a better understanding of immune regulation of the disease. Several investigators who have previously addressed this question reported that IFN- reduces MHC II expression on monocytes in vitro (9, 10), has antiproliferative effects on T cells (11, 12, 13), and inhibits T cell expression of matrix metalloproteinase 9, possibly impeding T cell migration across the blood-brain barrier (BBB) (14, 15, 16). IFN- has also been shown both to reduce the production of IL-12, which might inhibit the Th1 development, and to induce the production of IL-10 (17, 18, 19). All these mechanisms could be beneficial in a Th1-mediated disease. Nevertheless, despite tremendous efforts the mode of action of IFN- in MS patients is still unknown, and it has proven difficult to correlate many of the suggested mechanisms with the in vivo effects of this cytokine. In this study we further assess this question using IFN- gene-deleted (IFN- KO) mice and experimental autoimmune encephalomyelitis (EAE), the commonly used animal model for MS. Indeed, we observed augmented and chronic EAE with a higher incidence in IFN- KO mice. When trying to dissect the IFN--targeted pathway, we determined that the effect of IFN- is neither on priming of encephalitogenic T cells nor on B cell activation and Ab production. Moreover, deletion of the IFN- gene does not lead to any change in the Th1/Th2 balance, but, rather, leads to a generalized amplified inflammatory response in CNS with elevated levels of IFN-, IL-4, and TNF-. In addition, IFN- KO mice had significantly more activated microglia and macrophages than their wt littermates. We here suggest that the lack of IFN- leads to persistent activation of residual APCs in CNS, which results in prolonged inflammation and extensive demyelination.
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Materials and Methods |
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The IFN--deficient mice were originally developed with a neomycinR inserted targeting construct, containing Ig 
2 chain as a reporter gene, transfected to the 129 strain ES cell E14, and backcrossed to the C57BL/6 strain (20). Mice were subsequently backcrossed to the B10.RIII strain for seven generations and then were bred and kept at the conventional animal facility at Section for Medical Inflammation Research, Lund University. Eleven- to 18-wk-old, sex-matched littermates were used in all experiments. Throughout this study, both wild-type (wt) and heterozygous littermate mice were used to compare with the deficient KO mice. Since no discrepancy was observed between the heterozygous and homozygous wt groups, hereafter they will both be referred to as wt littermates.
PCR screening of mice
Tissue from tail tip or toes was collected for DNA preparation. Crude DNA preparation was made through tissue digestion with proteinase K and DNA precipitation with isopropanol. The animals were screened using PCR and a three-primer system: IFN- sense, 5'-TAT CTT CAG GGC TGT CTC CTT TCT-3'; IFN- antisense, 5'-ACC TGT TGT TCA TGA TGG AAG CCA-3'; and 2 antisense, 5'-GGC ATA GTT ACT AGT TGT AAC AGC-3' (MWG-Biotech, Risskov, Denmark). This PCR on IFN- KO and wt DNA produced fragments of different sizes; IFN- heterozygous mice presented both fragments.
Induction of active EAE and clinical evaluation
Myelin basic protein 89–101 (MBP89–101) peptide (VHFFKNIVTPRTP-COOH; Å. Engström, Uppsala University, Uppsala, Sweden) was used to induce EAE in IFN- KO and wt littermates. Mice were immunized s.c. at the base of the tail with 100 µl of emulsion of 250 µg of MBP89–101 peptide in PBS and CFA containing mycobacterium tuberculosis H37Ra (Difco, Detroit, MI). In addition, on the day of immunization and 2 days later, each animal received 400 ng of pertussis toxin (from Bordetella pertussis; Sigma-Aldrich, Stockholm, Sweden) dissolved in 100 µl of PBS i.p. The mice were weighed and examined three times a week between days 0 and 64. A scale ranging from 0–8 was used to assess the clinical severity of EAE: 0, no abnormalities; 1, tail weakness and a lowering of the hindquarter; 2, total paralysis of the tail; 3, mild waddle and total paralysis of the tail; 4, heavy waddle with impaired control of hind limbs; 5, paralysis of one of the hind limbs; 6, paralysis of both hind limbs; 7, paralysis of all limbs; and 8, death. Relapses were defined as improvement in clinical score after the acute phase of disease by at least two points on the scoring scale (remission phase), followed by a worsening of disease of a minimum of two points on the scoring scale. The accumulative score was calculated as the sum of all clinical scores of each individual mouse during the entire observation period and is thus equivalent to the area under the curve. The maximum clinical score was defined as the highest score that each individual reached during the whole experiment. All scorings were performed in a blind fashion.
T cell proliferation
Animals were sacrificed on days 10 and 64 postimmunization (p.i.), and lymph nodes and spleens were dissected out. Single-cell suspensions were prepared in PBS by passing through a sieve. Spleen cells were then treated with 0.84% NH4Cl to lyse RBC. Culturing medium consisted of DMEM with Glutamax I (Life Technologies, Stockholm, Sweden) supplemented with 10 mM HEPES, 50 µM 2-ME, 1% penicillin-streptomycin, and 5% heat-inactivated FCS (PAA Laboratories, Pasching, Austria). Cells were kept in an incubator at 37°C and 7.5% CO2. For measurement of Ag-induced T cell proliferation, cells were cultured in quadruplicate in round-bottom, 96-well plates (Nunc, Copenhagen, Denmark) at a concentration of 5 x 105 cells/well and were stimulated with MBP89–101 in concentrations ranging from 0 to 125 µg/ml. Purified protein derivative (10 µg/ml) was used as a positive control. Following 48 h of incubation, cells were pulsed with [3H]thymidine. After a further 24 h of incubation the cells were harvested on glass-fiber filters, and thymidine incorporation was measured in a beta scintillation counter (Matrix 96 Direct beta counter; Packard, Meriden, CT). The stimulation index was calculated by dividing Ag-specific proliferation by proliferation in medium only. Just before harvesting, supernatants were collected for analysis of cytokine content.
Spleen macrophage cultures
Mice were immunized for EAE as described above, and spleens were dissected out 9 days later. For cytokine studies, single-cell suspensions were prepared as described above, and cells were seeded into 24-well plates at a concentration of 20 x 106 cells/well. Cells were left to adhere for 90 min, whereafter the wells were flushed with cold medium to remove nonadherent cells. The plates were left for another 60 min and again washed. The cultures were then left untreated or were treated with either 10 U/ml of IFN- or 50 ng/ml LPS or first primed with 10 U/ml IFN- for 60 min then incubated with 50 ng/ml LPS. Supernatant were collected 3, 10, and 24 h later and assayed for cytokine content by ELISA. For FACS analysis, naive spleens were first partly digested with 1.6 mg/ml collagenase and 0.1% DNase for 30 min at 37°C. Digestion was stopped by adding 15% FCS, and single-cell suspensions were performed as described above. Thereafter, cells were washed twice in PBS containing 2% FCS. For enrichment of macrophages, cells were incubated for 20 min on ice with Abs against CD4 (H129), CD8 (53-6.7), and B220 (14.8); all were from BD PharMingen (San Diego, CA) and used at a concentration of 7.5 µg/ml. After washing to remove unbound Abs, Ab-bound cells were depleted using Dynabeads-conjugated mouse anti-rat IgG, following the manufacturer’s instructions (Dynal Biotech, Oslo, Norway). The macrophage-enriched cell suspensions were thereafter seeded in six-well plates at 5.5 x 106 cells/well and treated as described above.
FACS staining and evaluation
After 24 h in culture, macrophages were washed once with PBS and then removed from the culture plates using enzyme-free cell dissociation solution (Sigma-Aldrich) in combination with vigorous flushing with the pipette. After washing in FACS buffer (2% FCS in PBS), cells were first incubated with 10 µg/ml anti-FcR Ab (24.G.2, from our hybridoma collection) and thereafter incubated with Abs against Mac-1-FITC or PE (M1/70), ICAM-1-PE (3E2), ICAM-2-biotin (3C4), and VCAM-1-biotin (429) for 20 min on ice. All Abs were purchased from BD PharMingen and were used at 1–5 µg/ml. After washing, cells were incubated with streptavidin-allophycocyanin (BD PharMingen) diluted 1/400. The cells were analyzed using a four-color FACSort (BD Biosciences, Mountain View, CA). Cells that had a clear macrophage forward and side scatter distribution and were positive for Mac-1 were analyzed further. Positivity was evaluated using comparison with negative samples stained with irrelevant isotype-matched controls and streptavidin-allophycocyanin.
ELISA
The following primary Abs were used: anti-IFN- (5 µg/ml), anti-IL-4 (2 µg/ml); R6A2 and 11B11 (from our hybridoma collection), anti-TNF- (6 µg/ml); G281-2626 (BD PharMingen). The following secondary biotinylated Abs were used: anti-IFN- (0.6 µg/ml), anti-IL-4 (0.5 µg/ml); Ani8 and BVD6-24.G2 (from our hybridoma collection), anti-TNF- (1 µg/ml); MP6-XT3 (BD PharMingen). An Ab pair for IL-1 from R&D Systems (Minneapolis, MN) was used according to the manufacturer’s instructions.
FlouroNunc Maxisorp plates (Nunc) were coated with Abs, as described above, for 2 h at room temperature or at 4°C overnight. After washing, the plates were incubated with 10% FCS in PBS for 1 h to block unspecific binding. Thereafter, samples and recombinant cytokines (BD PharMingen) were added and incubated at 4°C overnight. Finally, the plates were incubated for 2 h at room temperature with secondary abs in PBS containing 10% FCS and 0.1% Tween. For detection, the plates were incubated with europium-avidin, followed by enhancement buffer according to the manufacturer’s instructions and then were measured for fluorescence intensity (Wallac Oy EG & G, Turku, Finland). The cytokine content in supernatants was determined when data were within the linear region of the standard curve calculated from levels of the recombinant cytokines.
Bioassay for IL-12
Cell culture plates were coated with anti-IL-12 Ab (C15.1; 2.5 µg/ml) and incubated at 37°C overnight. After washing, plates were incubated with 10% FCS in PBS for 1 h to block unspecific binding. Thereafter, supernatant from the macrophage cultures and control rIL-12 (BD PharMingen) were added in dilution steps of 1/2 and incubated at 37°C overnight. The plates were again washed, and then spleen cells from naive wt mice (prepared as described above) were added (106 cells/well). After 48 h of incubation the supernatants were removed and assayed for IL-12-induced IFN- production by ELISA as described above.
Passive transfer of EAE
IFN- KO and wt mice (eight per group) were immunized for EAE. On day 17 p.i. spleens and draining lymph nodes were dissected out, and single-cell suspensions were prepared as described above. Cells from each group were pooled, set to a concentration of 2.5 x 106 cells/ml, and stimulated with 2.7 µg/ml Con A. After 36 h of stimulation, cells were collected and washed three times in PBS. Thereafter, 40 x 106 living cells from each group were given i.v. to IFN- KO and wt mice (six per group). This gave a total of four groups, two IFN- KO groups and two wt groups receiving encephalitogenic T cells originated from IFN- KO or wt mice, respectively. On the same day and 2 days later each mouse received 400 ng of pertussis toxin i.p.
Histopathology
Mice were immunized for EAE and were sacrificed on days 12 and 64 p.i. Brains and spinal cords were dissected out. For immunohistochemistry, tissues were embedded in OCT compound (Sakura Finetek, Zoeterwoude, The Netherlands) and snap-frozen in isopentane on dry ice, tissues were then cryosectioned in 10-µm slices and kept at -70°C until staining. To investigate cell infiltration, the presence of macrophages (anti-Mac-1; M1/70; 3 µg/ml), APCs (anti-MHC II; 7.16.17; 1 µg/ml), B cells (anti-B220; 14.8; 5 µg/ml), and T cells was investigated (anti-CD3; 145.2C11; 2 µg/ml). For cytokine analysis, cells expressing IL-4, IFN-, TNF-, and IFN- were investigated using mAbs 11B11 (2 µg/ml), Ani-8 (5 µg/ml), XT22 (5 µg/ml), and 7F-D3 (0.5 µg/ml), respectively. Abs were obtained from BD PharMingen, except for 7F-D3, which was purchased from Sigma-Aldrich, and 11B11 and Ani-8, which were from our hybridoma collection. M1/70, 7.16.17, 11B11, and Ani-8 were directly biotinylated, whereas 14.8, 145.2C11, XT22, and 7F-D3 were detected using the secondary Abs, goat anti-Syrian hamster and goat anti-rat (Jackson ImmunoResearch Laboratories, West Grove, PA). ExtrAvidin-peroxidase (Sigma-Aldrich) and diaminobenzidine (50 mg/ml; Saveen Biotech, Malmö, Sweden) were used for detection, and hematoxylin or methyl green was used for background staining. When staining for cytokines, 0.1% saponin was used in all steps after fixation. In all studies the total numbers of positive cells was counted blindly in one brain section and one spinal cord section per mouse. Brain tissue, including medulla oblongata, was sampled by dividing the organ midsaggitally and sectioning parallel to the midline. Those sections in the immediate vicinity of the midline were used for measurement. The thoracic part of the spinal cords was collected and sectioned longitudinally; inner sections close to the central canal were used for evaluation. For demyelination studies, the lumbar part of the spinal cord was collected on day 64 p.i. These were fixed in 4% formalin in PBS for a minimum of 24 h. Thereafter, they were dehydrated, embedded in paraffin, and transversally sectioned at 6 µm. Sections were incubated for 6 h at 60°C in 0.1% Luxol Fast Blue for visualizing myelin. Lithium carbonate (0.05%) was thereafter used to destain nonmyelinated areas. Hematoxylin-erytrosin was used for nuclear counterstaining. Three transverse sections from each animal were analyzed using Easy Image Analysis 2000 (Bergström Instruments, Solna, Sweden). First the demyelination area in each section was measured, then the total spinal cord area was measured. From these three sections, a mean value for each mouse was calculated. Demyelination was expressed as square millimeters of demyelination per 100 mm2 total area of spinal cord section.
Ab response
Sera were collected from immunized mice on day 64 p.i. Semiquantitative ELISA determined the amounts of total anti-MBP and anti-myelin oligodendrocyte glycoprotein (MOG) IgG as well as IgG1 and IgG2a isotypes. The plates were coated overnight at 4°C with either MBP or MOG protein in PBS containing 10 µg/ml BSA (Sigma-Aldrich). Serum was then added in dilution steps of 10 and incubated for 2 h at room temperature. Biotinylated secondary Ab, namely, goat anti-mouse IgG1, goat anti-mouse IgG2a (Southern Biotechnology Associates, Birmingham, AL), or peroxidase-conjugated goat anti-mouse total IgG (Jackson ImmunoResearch Laboratories) was then added and incubated for 2 h at room temperature. Peroxidase-conjugated ExtraAvidin (Sigma-Aldrich) was added to the IgG1 and IgG2a plates and incubated for 1 h at room temperature. The plates were developed using the ABTS system (Roche, Mannheim, Germany) following the manufacturer’s instructions; thereafter, enzymatic activity was measured using colorimetric analysis at a wavelength of 405 nm.
Statistical analysis
Differences in clinical scoring were calculated using nonparametric Mann-Whitney testing. For the passive transfer, differences in accumulative score were first found significant using Friedman’s test and thereafter were further classified and compared pairwise using nonparametric Mann-Whitney testing. Disease incidence and relapse frequency were evaluated using the 
2 test. For T cell assays, FACS data, and cytokine and Ab ELISA, Student’s t test was used for statistical calculations. Histology was evaluated using nonparametric Mann-Whitney testing. A value of p < 0.05 was considered significant.
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Results |
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KO mice develop severe and chronic EAE with increased incidence compared with their wt littermates
As shown in Fig. 1, A and B, IFN- KO mice developed significantly more severe and chronic EAE with higher susceptibility to disease compared with their wt littermates. The data presented here represent a pool of four separate experiments, since there were no discrepancies detected between experiments. It is noteworthy that endogenous IFN- did not affect the onset of disease nor did it significantly alter the severity of EAE in the acute phase, but, rather, predominantly effected chronicity. Many of the affected wt animals recovered after the initial paralysis, and only 40% had clinical symptoms at the end of the experiment. In contrast, the majority of affected IFN- KO mice continued to increase in disease severity, developing chronic disease or suffering relapses; thus by day 64 90% of the IFN- KO mice still had EAE (Fig. 1B). During the chronic phase of the disease, the mean score and incidence as well as the relapse rate were higher in IFN--deficient mice than in wt animals (Fig. 1, A and B, and Table I). Disease severity, calculated either as mean maximal score or as accumulative score, was also significantly higher in IFN- KO mice compared with their wt littermates (see Table I). Fig. 1C illustrates disease occurrence in five individual wt mice to exemplify different EAE patterns and relapses.
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To investigate whether an enhanced expansion of autoreactive T cells could be responsible for the observed differences in EAE, T cells were rechallenged with MBP89–101 in vitro on day 10 p.i. No differences were observed between IFN- KO and wt mice regarding the capacity of T cells to proliferate or produce IFN- (Fig. 2, A and B) and IL-4 (data not shown) in response to recall Ag. These findings argue against any influence of IFN- on priming of autoreactive T cells. Moreover, our data do not support any role for IFN- in affecting the balance between Th1 and Th2 responses in a disease well known to be Th1 mediated.
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KO and wt mice are equally encephalitogenic
To further investigate whether the difference in EAE susceptibility and severity between IFN- KO and wt mice lies in the priming of encephalitogenic T cells, Con A-stimulated spleen and lymph node cells from immunized IFN- KO or wt mice were transferred to either IFN- KO or wt naive mice. Interestingly, we observed that IFN- KO recipient mice developed significantly more severe EAE with a higher incidence compared with wt mice regardless of the origin of encephalitogenic T cells (Fig. 3 and Table II). Note that incidence in Fig. 3 refers to the percentage of mice that were simultaneously affected at any given time point, while incidence in Table II refers to the number of mice that developed EAE during the observation period. This gives further support to the idea that IFN- does not affect the priming of autoreactive T cells, nor does it affect the capacity of expanded cells to be more encephalitogenic due to higher proinflammatory cytokine production.
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KO and wt mice
We next investigated whether differences in B cell activation and autoantibody production could explain the difference in neurological symptoms of EAE between IFN- KO and wt mice. Our data could not confirm any suspicion of differential activation of B cells as measured by autoantibody production. No difference in MBP-specific total IgG or IgG2a was observed (Fig. 4, A and B), and no MBP-specific IgG1 could be detected in either of the groups (data not shown). Moreover, we hypothesized that tissue damage in the CNS could lead to endogenous release of other myelin Ags, e.g., MOG, and this might result in different autoantibody production, possibly explaining the difference in clinical symptoms in the chronic phase. However, we could not detect any differences in MOG-specific IgG between the groups (data not shown).
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leads to augmented macrophage activation
Since we did not detect any distinction in encephalitogenic T cells or autoantibody production by B cells between the two groups, we subsequently examined whether there were any differences in macrophage capacity to produce proinflammatory cytokines and/or in macrophage expression of molecules associated with adhesion and activation. Thus, macrophage cultures from IFN- KO and wt mice were analyzed for TNF-, IL-1, and IL-12 during different time points after LPS or a combination of IFN- and LPS stimulation in vitro. Maximum cytokine responses were detected in cultures treated with both IFN- and LPS. Since similar data were obtained in cultures stimulated with LPS only, we have only shown the IFN- and LPS stimulation results. The highest levels for IL-1 and IL-12 were measured after 24 h; however, there were no differences observed between IFN- KO and wt mice (Fig. 5, A and B). Interestingly, in wt mice TNF- levels reached a peak after 10 h and then slowly declined over 24 h (Fig. 5C). This was in sharp contrast to the IFN- KO mice, in which TNF- levels continued to increase well over the wt levels without reaching a plateau (Fig. 5C). This implies that IFN- is a natural regulator of macrophage activation and TNF- production in wt mice, but is disrupted in IFN- KO mice. Indeed, IFN- has been shown to be induced in macrophages upon LPS stimulation (21). Subsequently, we evaluated macrophage activation by analyzing the expression of ICAM-1, ICAM-2, VCAM-1, and Mac-1 in cells cultured for 24 h with or without IFN- and LPS stimulation. In accordance with previous observations (22, 23), we found an up-regulation of ICAM-1 and Mac-1 after IFN- plus LPS stimulation (Fig. 5D). ICAM-1 expression was similar in wt and IFN- KO mice, but IFN- KO mice had a significantly higher expression of Mac-1 after activation. ICAM-2 expression was not significantly affected by stimulation. IFN- KO mice expressed slightly less ICAM-2 before stimulation, but this difference was no longer significant in IFN-- and LPS-activated macrophages. VCAM-1 expression decreased after stimulation, and again this decrease was significantly more pronounced in IFN- KO mice compared with wt mice (Fig. 5D). Taken together these data suggest that IFN- limits macrophage activation at several levels determined by Mac-1, VCAM-1 expression, and TNF- production.
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KO mice have extensive and persistent inflammation in CNS without association with a skewed Th1/Th2 phenotype
Animals were sacrificed during the onset and chronic phase of EAE and were analyzed for the degree and type of inflammatory cells infiltrating the CNS and what cytokines were produced in situ. We observed that IFN- KO mice have significantly more infiltrating T cells, activated macrophages, and MHC II-expressing cells during the disease acute phase (day 12 p.i.) compared with the wt group (Fig. 6A). During the chronic phase (day 64 p.i.), the only difference detected in terms of cell types in CNS was a significantly higher number of Mac-1-expressing cells in IFN- KO mice compared with wt animals. The majority of these cells displayed the typical morphology of activated residual microglia and were scattered throughout the CNS parenchyma (Fig. 6, B and C). However, there was a more pronounced CNS inflammation in IFN- KO mice, as measured by the elevated number of cells producing both the Th1 cytokines IFN- and TNF- as well as the Th2 cytokine IL-4 (Fig. 7, A–H). These findings demonstrate that the lack of IFN- does not eventually lead to a Th1-skewed cytokine response but, rather, augments a generalized inflammatory effect in the CNS. Notably, the cytokine showing the greatest difference in wt mice was TNF-. This is not a typical T cell cytokine, but can also be produced by resident APCs in the CNS. Indeed, we observed TNF--positive cells mainly among infiltrating macrophages and residual microglia. These data once more indicate that there may be a dysregulation of inflammation in the CNS, rather than an effect on T cell priming in the periphery.
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KO mice develop widespread demyelination in the CNS
Mice were sacrificed on day 64 p.i. and examined for the extension of demyelination. As depicted in Fig. 8, A–C, we detected a significantly higher degree of demyelination in IFN- KO mice compared with wt littermates. This finding correlated well with the clinical findings as well as with the general scale of inflammation in the CNS.
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is produced in the CNS of EAE-affected wt mice
We continued to investigate whether the differences in microglia activation and cytokine production in the CNS, with consecutive distinction in clinical symptoms between wt and IFN- KO mice, were associated with regulation of IFN- production at the site of inflammation. Accordingly, we investigated whether IFN- was produced locally in the CNS. Only in wt mice did we find a massive increase in IFN--producing cells in the CNS during the acute phase of the disease (Fig. 9A). These cells were present both in perivascular infiltrates as well as in cells bearing the morphology of ramified microglia (Fig. 9, B and C). IFN--positive cells were still present in the CNS at the end of the experiment (Fig. 9A), although in lower numbers, and at this time they consisted almost entirely of microglia-like cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment is the prevention of relapses (7, 8). This is in agreement with observations in this report, in which we used MBP89–101 to induce EAE in mice as a model for MS. Our study revealed that IFN- gene deletion resulted in an increase in the frequency of relapses, with more animals developing augmented and chronic disease.
EAE is a Th1-mediated disease, and it has been reported that IFN- can both limit T cell proliferation (24) and inhibit Th1 development in vitro (25, 26). However, in our study the capacity of T cells to proliferate in vitro after immunization for EAE was unaltered when IFN- was absent. Furthermore, we could not detect any difference in the effector function of T cells, since MBP89–101-specific T cells from wt and IFN- KO mice produced equivalent amounts of IFN- and IL-4. Hence, our findings do not support the idea that IFN- may act beneficially either through limiting T cell proliferation or through skewing the T cell response from Th1 to Th2. To further investigate this, we used a passive transfer model of EAE by transferring autoreactive T cells. This demonstrated that primed T cells from IFN- KO and wt mice were equally encephalitogenic and that the lack of IFN- in the recipients was the decisive factor for the development of an augmented EAE. Together these data suggest that endogenous IFN- does not affect the primary activation of encephalitogenic T cells in peripheral lymphoid organs and hence does not influence the initiation phase of the disease. Nevertheless, one could assume that IFN- can influence T cell effector functions after the initial activation, as it has been reported that IFN- inhibits matrix metalloproteinase-9 in T cells cultured in vitro (15, 16) and down-regulates very late Ag-4 (VLA-4) on lymphocytes from MS patients (27), and this could impair T cell ability to cross the BBB (28, 29). Our observation of activation-induced down-regulation of VCAM-1 observed on the in vitro cultured macrophages could be important for cell adhesion and migration. Others have reported no effect of LPS on macrophage VCAM-1 expression, but, rather, an increase in VCAM-1 expression on endothelial cells in response to various inflammatory stimuli (30, 31, 32). VCAM-1 expression on endothelial cells serves as an adhesion molecule and binds VLA-4-expressing cells, mainly monocytes, macrophages, and activated T cells. Numerous studies have shown the importance of an interaction between VLA-4+ lymphocytes and VCAM-1+ endothelial cells for the development of EAE (33, 34, 35). In this context, down-regulation of VCAM-1 on macrophages could promote VLA-4+ T cell interaction with VCAM-1+ endothelial cells and subsequent transendothelial migration. However, whether our observation of down-regulation of VCAM-1 on macrophages has relevance in vivo in this model is unclear. Even though we detected extensive CNS cell infiltration in IFN- KO mice during acute phase of EAE, it is unlikely that the capacity of T cells to pass the BBB in the absence of endogenous IFN- is a decisive factor. The reasons we conclude this are as follows. Firstly, the clinical differences are observed in the chronic phase of the disease where T cell infiltration was similar in wt and IFN- KO mice. Secondly, the passive transfer of EAE clearly established that T cells from wt and IFN- KO mice exhibit equal encephalitogenic ability, suggesting similar capacities to infiltrate the CNS.
Myelin-specific autoantibodies have been postulated to enhance demyelination and sustain chronic inflammation in the CNS (36, 37, 38). It has also been claimed that IFN- influences the production of self-reactive Abs (39). Yet, our analysis of autoantibody production indicated that differences in clinical symptoms of disease between wt and IFN- KO mice could not be explained by a discrepancy in autoantibody production, as the myelin-specific IgG and IgG2a titers did not differ between the two groups.
Interestingly, histopathological analysis revealed a much more extensive inflammation in CNS in IFN--deficient mice in both acute and chronic phases of EAE, with more cells producing both IFN- and IL-4. While inflammation during the acute phase was generalized, characterized by infiltration of T cells and activated MHC class II-positive macrophages, the chronic phase CNS inflammation was very distinct. To our surprise, we could no longer detect differences in infiltrating cell numbers. However, a large number of highly activated Mac-1 expressing residual microglial cells was scattered throughout the CNS parenchyma in the IFN- KO group, and this was virtually absent in the wt group. Mac-1, also referred to as CD11b/CD18 or CR3, is constitutively expressed on cells of the myeloid macrophage lineage, but is further up-regulated upon activation (40, 41). It can bind to several receptors and molecules, such as ICAM-1/2, C3bi, and fibrinogen (42). Ligation of Mac-1 results in cytoskeleton rearrangement, increased phagocytic capacity, and production of several proinflammatory mediators, such as IL-1, IL-6, TNF-, and NO (42, 43, 44, 45, 46). Abs blocking Mac-1 have been shown to inhibit myelin phagocytosis in vitro (47) and reduce clinical signs of EAE (48). In addition, it has been shown that in vivo activated Mac-1+ microglia from EAE-affected mice are much more efficient APCs than counterparts from naive mice (49).
We also observed that ramified microglia cells were highly positive for TNF-. It is worth pointing out that TNF- was the prominent cytokine that differed in the two groups during the chronic phase of EAE. This gives further support to our in vitro findings that macrophages from IFN- KO mice both expressed elevated levels of Mac-1 after stimulation and produced an increasing amount of TNF-, while in the wt group TNF- production was limited. The important role for TNF- in creating pathogenic tissue damage has been demonstrated in several studies in which inhibiting this cytokine using Abs, soluble receptors, or by eliminating macrophages ameliorated clinical symptoms of EAE (50, 51, 52). Here we report that endogenous IFN- inhibits full expression of Mac-1 as well as selectively inhibits TNF- production, but not other macrophage-specific cytokines, such as IL-12 and IL-1. The increased TNF- production could be secondary to elevated Mac-1 expression, since stimulation through Mac-1 has been shown to result in TNF- release (43, 45) Furthermore, we show for the first time that inflammation alone is sufficient as a triggering factor for IFN- production in the CNS. Thus, it acts as a limiting factor for activation of and TNF- production by macrophages and residual microglia in the CNS. In agreement, it has been reported that the inflammatory graft-vs-host response could trigger IFN- production by macrophages (53).
This study demonstrated the effects of endogenous IFN- under more physiological conditions, which could in part explain the discrepancies of reported in vitro results. We conclude that the greater severity and chronicity of EAE in IFN- KO mice are not due to its effect on T cell priming or the encephalitogenicity of T cells, nor does it affect the balance between Th1 and Th2 responses. In summary, our data suggest that the locally produced IFN- serves to limit macrophage and microglia Mac-1 expression and activation, which inhibit local TNF- production. We further postulate that limiting these potential APCs reduces the in situ T cell reactivation and cytokine production (IFN- and IL-4) and thus breaks the inflammatory spiral that otherwise leads to chronicity and tissue degeneration. This regulatory factor is absent in IFN- KO mice, and the outcome is a sustained chronic inflammation leading to more demyelination and consequent neurological deficits.
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2 Address correspondence and reprint requests to Dr. Shohreh Issazadeh-Navikas, Section for Medical Inflammation Research, Institute for Cell and Molecular Biology, University of Lund, I11, BMC, 221 84 Lund, Sweden. E-mail address: shohreh.issazadeh{at}inflam.lu.se
3 Abbreviations used in this paper: MS, multiple sclerosis; BBB, blood-brain barrier; EAE, experimental autoimmune encephalomyelitis; KO, knockout; p.i., postimmunization; MBP, myelin basic protein; VLA-4, very late Ag-4; wt, wild type; MOG, myelin oligodendrocyte glycoprotein.
Received for publication October 7, 2002. Accepted for publication February 28, 2003.
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