Short-lived immunization site inflammation in self-limited active experimental allergic encephalomyelitis

Francesca Di Rosa1,5, Barbara Serafini3, Paola Scognamiglio1,6, Antonio Di Virgilio4, Luigi Finocchi1, Francesca Aloisi3 and Vincenzo Barnaba1,2

1 Fondazione Andrea Cesalpino, Istituto I Clinica Medica, Università `La Sapienza', Rome 00161, Italy
2 Istituto Pasteur-Cenci Bolognetti, Rome 00185, Italy
3 Laboratorio di Fisiopatologia di Organo e di Sistema and
4 Servizio Qualità e Sicurezza Sperimentazione Animale, Istituto Superiore di Sanità, Rome 00161, Italy

Correspondence to: V. Barnaba, Fondazione Andrea Cesalpino, Istituto I Clinica Medica, Università di Roma `La Sapienza', Rome 00161, Italy


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
To understand the mechanisms underlying spontaneous remission of proteolipid protein (PLP) 139–151 peptide-induced experimental allergic encephalomyelitis (EAE), an acute autoimmune disease of SJL mice resembling human multiple sclerosis, we examined both the effector response site in the central nervous system (CNS) and the immunization site at different phases of the disease. In the CNS, the frequency of PLP 139–151 peptide-specific IFN-{gamma}-producing T cells as well as the amount of infiltrating CD4+ and CD11b+ cells decreased with recovery. However, IL-4-producing cells were always rare and cyclooxygenase-2+ cells were numerous only at disease peak in the CNS, suggesting that Th2 cytokines and prostaglandins did not determine remission of EAE. By looking at the s.c. site of PLP 139–151 peptide plus adjuvant injection, we found that, although the inflammatory infiltrate was abundant, CD11b+ cells started to decrease already during disease acute phase and DEC-205+ cells were numerous only at early time points. We propose that immunization site inflammation is short-lived in PLP 139–151 peptide-induced EAE, and this leads to a temporary autoreactive T cell stimulation and to a self-limited disease.

Keywords: adjuvant, autoimmunity, immunization, inflammation, T cell cytokines


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
Experimental allergic encephalomyelitis (EAE) is a Th1-mediated autoimmune disease, which resembles human multiple sclerosis (MS) and can be induced in the mouse with either active immunization with central nervous system (CNS) antigens or passive transfer of T cells specific for myelin-derived peptides (1,2). In both cases, effector CD4+ T cells specific for myelin components migrate to the CNS, recognize their target antigen there and release Th1 cytokines, causing inflammation and tissue damage. Diseased mice experience weight loss, weakness and neurological symptoms. Depending both on genetic factors and immunization protocol, EAE can have either an acute or chronic relapsing-remitting clinical course.

The mechanisms leading to a self-limited disease in the case of acute EAE are still unclear, although several factors have been involved in autoreactive response inhibition, including TCR Vß-specific suppressor T cells, Th2 and/or other T cell regulatory cytokines, corticosteroid hormones, and prostaglandins (PG) (37). Even if anti-inflammatory Th2 cytokines have often been involved in the inhibition of pathogenic Th1 cells in autoimmune diseases (4,8,9), we have recently shown that IL-4-producing cells do not play a role in the termination of the acute phase of proteolipid protein (PLP) 139–151 peptide-induced EAE in SJL mice (10).

Here, we examined some putative regulatory mechanisms involved in spontaneous recovery of the acute phase of PLP 139–151 peptide induced EAE in SJL mice and tested the hypothesis that EAE remission is dependent on the termination of the acute inflammation induced by exogenous antigen plus adjuvant injection.

First, we re-examined the question of Th1/Th2 balance in PLP 139–151 peptide-induced EAE by using ELISPOT, a very sensitive technique, especially for detecting a few IL-4-producing cells (11), which allowed us to check our previous results obtained by ELISA (10). Indeed, in contrast to ELISA determining only the Th1/Th2 profile of the T cells, ELISPOT allowed us to measure the frequencies of PLP 139–151-specific IFN-{gamma} and IL-4-producing T cells in the CNS infiltrate of SJL mice in different phases of disease, and to correlate these numbers with the amount and the localization of CNS-infiltrating CD4+ and CD11b+ cells, as detected by immunohistochemistry.

Second, we investigated whether some PG play a regulatory role in EAE, as previously proposed (7). PG, the arachidonic metabolites of the cyclooxygenase (COX) enzyme pathway, comprise a family of local mediators with a major role in regulating inflammation, immune responses and neurotransmission. Besides the constitutive isoform COX-1, a second inducible isoform, termed COX-2, is expressed by inflammatory cells and is responsible for the production of high levels of PG. Although COX-2 is classified as a pro-inflammatory enzyme, some PG, like PGD derivatives and PGE2, were recently reported to have anti-inflammatory function (12,13 and references therein). To evaluate the involvement of PG both in acute inflammation stage and in recovery, we analyzed COX-2 expression in the CNS of SJL mice in different phases of EAE by immunohistochemical methods.

Finally, we considered the possibility that the key element leading to a self-limited disease might reside in the site of anti-PLP 139–151 response induction. To get insight into the first steps of the cascade leading to autoreactive T cell activation, we performed an immunohistochemical analysis of the s.c. site of peptide and adjuvant injection at different times during the clinical course of EAE and correlated the amount of inflammatory cells infiltrating the s.c. tissue with the changes observed in the CNS.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
Animals
SJL female mice were purchased from Charles River (Calco, Italy), housed at the Animal Facility of the Istituto Superiore di Sanità and used at 8–12 weeks of age.

Active EAE protocol
The peptide corresponding to amino acids 139–151 (HSLGKWLGHPDKF) of mouse PLP was synthesized as described (10). We injected the mice with 0.2 mg PLP 139–151 peptide in complete Freund's adjuvant (CFA; Difco, Detroit, MI) s.c. in the flank, on day 0 and 7, and with 200 ng pertussis toxin (Sigma, St Louis, MO) i.p., on day 0, 1, 7 and 8. Control mice were treated with PBS in CFA, plus pertussis toxin, according to the same schedule. Mice were observed till day of sacrifice (day 17–50) and scored according to their clinical severity as reported (10).

ELISPOT
After isolation (10), lymph node cells (1,000,000 and 500,000 cells/well) and CNS infiltrating cells (10,000 cells/well) of pooled PBS and PLP 139–151 peptide-treated mice were cultured in MAHA nitrocellulose microtiter plates (Millipore, Bedford, MA), which had been precoated either with anti-IFN-{gamma} or anti-IL-4 mAb (Endogen, Woburn, MA) (14). Cells were cultured overnight at 37°C with 5% CO2 in 0.1 ml of 10% FCS RPMI medium (Life Technologies, Paisley, UK) supplemented with glutamine, penicillin, streptomycin, non-essential amino acids, sodium pyruvate and ß-mercaptoethanol, in the presence of medium alone or 50 µg/ml of PLP 139–151 peptide. Plates were extensively washed with 0.05% Tween 20 PBS and then incubated with the second corresponding biotinylated mAb (biotinylated anti-IFN-{gamma} or anti-IL-4 mAb, Endogen) for 5 h at room temperature. After further washing, plates were incubated with poli-peroxidase-labeled streptavidin (Poli-HRP-40-SA; Research Diagnostics, Flanders, NJ) for 30 min at room temperature. Spots were developed with freshly prepared substrate solution (0.3 mg/ml 3-amino-9-ethylcarbazole and 0.015% H2O2 in 0.1 M sodium acetate pH 5) and counted using a Leitz stereomicroscope.

Immunohistochemistry
For specimen collection, mice were anesthetized with xylazine chloridrate and ketamine, and perfused with PBS followed by cold 4% paraformaldehyde PBS intracardiacally. CNS and some cutaneous samples corresponding to the sites of immunization were then removed. Tissues were kept over- night in 4% paraformaldehyde PBS at 4°C, passed in 15 and 30% sucrose PBS, frozen in dry ice-chilled isopentane, cut with cryostat, and stored at –20°C. For staining, 10 µm thick sections were dried, passed in 70, 95 and 100% ethanol, and then dried again. After rehydratation with PBS and pre-incubation with 10% normal rabbit serum, sections were incubated with one of the following primary antibodies: rat anti-mouse CD4 (RM4-5; PharMingen, San Diego, CA), rat anti-mouse CD8 (53-6.7, PharMingen), rat anti-mouse CD11b (M1/70, Boehringer Mannheim, Germany), goat anti-rat COX-2 (M-19; Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-mouse DEC-205 (NLDC-145; Serotec, Raleigh, NC). After extensive washing with PBS, sections were incubated with the corresponding biotinylated secondary antibodies, either rabbit anti-rat IgG or rabbit anti-goat IgG (Vector, Burlingame, CA). This was followed by a revelation step, with ABC peroxidase (Vectastain kit; Vector) and diaminobenzidine substrate, and a counterstaining procedure with hematoxylin. To eliminate endogenous peroxidase activity, sections were incubated with 0.3% H2O2 PBS before secondary mAb addition. Sections were observed under an Axiophote Zeiss microscope. The amount of positive cells was judged semiquantitatively by a blind observer according to the following scale: – = negative staining; ± = trace labelling; + = <10 positive cells per section; ++ = 10–50 positive cells per section; +++ = 50–200 positive cells per section; ++++ = >200 positive cells per section.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
Analysis of IFN- {gamma}- and IL-4-producing cells in the CNS of SJL mice at different stages of PLP 139–151 peptide-induced EAE
We injected SJL female mice s.c. with PLP 139–151 peptide in CFA and i.p. with pertussis toxin to induce EAE. The great majority of the treated mice showed signs of acute EAE, with clinical grade ranging between 2 and 4, whereas control mice injected with PBS in CFA and pertussis toxin were asymptomatic. As shown in Fig. 1(A)Go, PLP 139–151 peptide-induced EAE peaked around day 16–17 after immunization, spontaneously resolved in a few days, and did not relapse. At day 17 after immunization, during disease acute phase, and at day 26 and 35, during remission, we isolated CNS-infiltrating cells, and measured by ELISPOT the frequency of IFN-{gamma}- and IL-4-producing cells in the infiltrate, after an overnight culture with medium alone or PLP 139–151 peptide. As recently demonstrated, ELISPOT assay allows enumeration of antigen-specific T cells with a sensitivity similar to the MHC–peptide tetramer technique (15). Figure 1(B)Go summarizes the results of seven of these experiments performed with a total of 26 mice. The CNS-infiltrating cell cytokine profile is in agreement with that obtained by ELISA and reported in our previous study on a group of 70 PLP 139–151 peptide-treated mice (10). IFN-{gamma}-producing cells were present in the CNS infiltrate at each time point tested, confirming our previous results showing that IFN-{gamma} production decreased very slowly in the CNS of diseased mice following clinical recovery and returned to baseline around day 77 after immunization (10). By using ELISPOT, we were able to detect IFN-{gamma} release by CNS-infiltrating cells even in cultures with medium alone, suggesting that some cells were actively producing this cytokine in vivo and continued to do so without any further stimulation. At any time point, the average frequency of IFN-{gamma}-producing cells in the CNS infiltrate was higher in the cultures with PLP 139–151 peptide than in those with medium alone. However, the PLP 139–151 peptide-specific IFN-{gamma}-producing T cell frequency was not very high even during disease acute phase, supporting the view that a major component of the CNS infiltrate was contributed by activated T cells aspecifically recruited to the CNS and stimulated via bystander mechanisms (1618). The average frequency of T cells specifically releasing IFN-{gamma} in response to PLP 139–151 peptide decreased with time during remission, in agreement with our previous results (10). IL-4-producing cells in the CNS infiltrate were very few or undetectable during both disease acute phase and remission, in line with the results we obtained by measuring IL-4 released in the cell culture supernatants by ELISA (10) and ruling out the possibility that a few Th2 cells producing small amounts of IL-4 in the CNS infiltrate appeared upon recovery. We also measured by ELISPOT the frequency of IFN-{gamma}- and IL-4-producing T cells in the PLP 139–151 peptide-stimulated cultures of lymph node cells from EAE diseased mice and found that IFN-{gamma}-producing cells ranged from 1 to 17 in 106 and IL-4-producing cells from 3 to 10 in 106. Both IFN-{gamma}- and IL-4-producing cells were still detectable in cultures of lymph node cells from PLP 139–151 peptide-treated mice at late remission time points, confirming our previous findings (10). Thus, Th1 cells are preferentially recruited to the inflamed CNS, in agreement with the described homing pattern of Th1 and Th2 cells (19).



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Fig. 1. Disease course of PLP 139–151 peptide-induced EAE and frequency of IFN-{gamma}- and IL-4-producing cells in the CNS infiltrate at different time points. (A) Time course of the clinical score of one typical EAE diseased mouse and one PBS-treated control. (B) Frequency of IFN-{gamma}- and IL-4-producing cells in the CNS infiltrate. At day 17, 26 and 35 after immunization we isolated CNS-infiltrating cells from PLP 139–151 peptide-treated mice, and measured the frequency of IFN-{gamma}- and IL-4-producing cells by ELISPOT, after an overnight culture in the presence of medium alone or 50 µg/ml of PLP 139–151 peptide. Each symbol represents a single mouse or a pool of two mice. The clinical score of the PLP 139–151 peptide treated mice ranged from 2 to 3 at day 17, and was 0 at day 26 and 35. The average frequency of cells specifically releasing IFN-{gamma} and IL-4 in response to PLP 139–151 peptide was calculated at any time point and is shown with a dotted line. In similar cultures of cells from PBS-treated control mice, IFN-{gamma}- and IL-4-producing cells ranged from 0 to 5 (in 105).

 
Taken together, our data confirm that clinical recovery of EAE is associated with a decrease of CNS-infiltrating Th1 cells and that Th2 regulatory cells are not involved in spontaneous remission (10), in line with other reports questioning the protective role of Th2 cells in autoimmunity (20, 21). We already showed that IL-4-producing cells specific for antigens other than PLP 139–151 peptide are not recruited to the CNS during recovery of PLP 139–151 peptide-induced EAE, as well as that regulatory T cells producing IL-10 and transforming growth factor-ß are not involved in remission of disease (10). The possibility that TCR Vß-specific suppressor T cells contribute to pathogenic T cell inhibition is also very unlikely in PLP 139–151 peptide-induced EAE in SJL mice, given the heterogeneous TCR repertoire of the encephalitogenic T cells (22). Collectively, these findings suggest that regulatory T cell circuits do not play a major role in pathogenic T cell inhibition in the CNS of diseased SJL mice recovering from PLP 139–151 peptide-induced EAE.

Analysis of CD4+, CD11b+ and COX-2+ cells in the CNS of SJL mice at different stages of PLP 139–151 peptide-induced EAE
To follow the pattern of CNS inflammation, we performed immunohistochemical analysis of the CNS of SJL mice at different time points: at day 10 after immunization, before any clinical evidence of disease; at day 16, during the acute phase; and at days 22, 28 and 40, during remission. Data are summarized in Table 1Go and some representative photomicrographs are shown in Fig. 2Go. After staining of the CNS with anti-CD4 and anti-CD11b mAb, only CD11b+ intraparenchymal microglia were detected at day 10 in PLP 139–151-treated mice (data not shown), whereas numerous CD4+ and CD11b+ cells were present at day 16, in agreement with the reported pattern of EAE inflammatory infiltrate (23). During the acute phase, the CD4+ and CD11b+ cells infiltrated the CNS parenchyma of spinal cord, brainstem and cerebellum. Infiltrating CD11b+ and CD4+ cells slowly decreased with clinical recovery, and almost completely disappeared from the CNS at day 40. Since day 28 of remission, the infiltrating CD4+ and CD11b+ cells were detectable only around blood vessels and inside the meninges, and just inside the meninges at day 40. CD8+ cells were rare at any tested time point (data not shown). The disappearance of the intraparenchymal inflammatory infiltrate during early remission correlates with clinical recovery and with the decrease of the PLP 139–151 peptide-specific IFN-{gamma}-producing T cell frequency in the CNS (Fig. 1Go). The persistence of perivascular and intrameningeal leukocytes during remission is consistent with the detection of some IFN-{gamma}-producing T cells in the CNS infiltrate even at day 35 by ELISPOT (Fig. 1Go). Such Th1 cells could fail to give overt neurological symptoms due to their localization. PBS-treated control mice did not show any infiltrating CD4+ and CD11b+ cells in the CNS (data not shown).


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Table 1. CD4+, CD11b+ and COX-2+ cells in the CNS of SJL mice at different phases of PLP 139–151 peptide-induced EAE
 


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Fig. 2. Photomicrographs of stained sections from the CNS of PLP 139–151 peptide-treated mice at day 16 and 22 after immunization. Representative panels from spinal cord sections are shown (x350 magnification for panel b and x500 for the others). (a and b) Anti-CD4 mAb; (c and d) anti-CD11b mAb; (e and f) anti-COX-2 antibody. Panels (c) and (e) are adjacent sections. (a, c, e) Day 16; (b, d, f) day 22.

 
To elucidate the possible inhibitory role of other factors in the CNS of EAE recovering mice, we looked at the expression of COX-2 enzyme during different phases of disease. In the acute carrageenin-induced inflammation COX-2 peaks twice: the first peak at 2 h is mainly associated with PGE2 synthesis and with pro-inflammatory activity, the second one at 48 h is related with PGD2 and 15deoxy{triangleup}12–14 PGJ2 production and anti-inflammatory function (24). In the EAE model the role of COX-2 is not clear yet and it has been suggested that PGE2 produced during the remission phase can down-regulate disease (7), consistent with its inhibitory effects on macrophage and Th1 cell function (13). Indeed, the finding that Th1 cells stimulate PGE2 production by microglia suggests that T cell-dependent production of PG may contribute to negative feedback mechanisms limiting inflammation in the CNS (25). We stained tissue samples from the CNS of SJL mice at day 10, 16, 22, 28 and 40 after immunization with anti-COX-2 antibody, and found high levels of positivity in the cellular infiltrate only at day 16, during disease acute phase. Staining of adjacent sections demonstrated that COX-2 was mainly expressed by infiltrating CD11b+ cells (Fig. 2c and eGo). COX-2 expression was reduced at day 22, during early remission, being weakly expressed by scattered intraparenchymal cells at this and late time points (Table 1Go and Fig. 2Go). COX-2+ cells were undetectable in the CNS of PBS-treated control mice (data not shown). Our results show that COX-2 expression in the CNS during the course of EAE is associated with the presence of the leukocyte infiltrate during disease acute phase. Although we cannot rule out that some of the PG produced in the CNS have an anti-inflammatory effect and contribute to local inhibition of Th1 cells and macrophages (13), termination of EAE does not appear to correlate with an increase of COX-2 expression during remission.

Analysis of CD4+, CD11b+ and DEC-205+ cells in the site of antigen injection of SJL mice at different stages of PLP 139–151 peptide-induced EAE
We then analyzed the cellular infiltrate in the s.c. site of antigen injection in SJL mice at day 10, 16, 22 and 40 after immunization (Table 2Go and Fig. 3Go). We observed that CD4+ cells were present in the s.c. tissue of PLP 139–151 peptide plus CFA-treated mice at any time point tested and reached their maximal level at day 16, whereas CD11b+ cells peaked at day 10 and started to decrease already at day 16, at the onset of the acute phase. Since day 16, a portion of CD11b+ cells in the s.c. tissue showed morphological signs of apoptosis (Fig. 3Go). Both CD4+ and CD11b+ cells infiltrated the s.c. tissue of PBS plus CFA-treated mice with similar distribution and kinetics, but reduced numbers, compared to the corresponding population in PLP 139–151 peptide plus CFA-treated mice. Untreated mice did not show any CD4+ and CD11b+ cells in the s.c. tissue (data not shown).


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Table 2. CD4+, CD11b+ and NLDC-145+ cells in the s.c. site of immunization of SJL mice at different phases of PLP 139–151 peptide-induced EAE
 


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Fig. 3. Photomicrographs of stained sections from the s.c tissue of PLP 139–151 peptide-treated mice at day 10 and 16 after immunization. Representative panels from s.c. tissue sections are shown (x500 magnification). (a and b) Anti-CD4 mAb; (c and d) anti-CD11b mAb; (e and f) anti-DEC-205 mAb. (a, c, e) Day 10; (b, d, f) day 16.

 
We also stained s.c. tissue sections with NLDC-145 mAb, specific for the dendritic cell marker DEC-205, and found a high level of positivity in PLP 139–151 peptide plus CFA-treated mice at day 10 and 16 after immunization. Dendritic cell-shaped DEC-205+ cells were preferentially localized in the outer skin layers at day 10 after immunization, were present throughout skin and s.c. tissue at day 16, and started to decrease at day 22, when some of them showed morphological signs of apoptosis (Table 2Go and Fig. 3Go). In PBS plus CFA-treated mice some DEC-205+ cells with dendritic morphology were present in the outer skin layers at day 10 after immunization. PBS plus CFA-treated mice at day 16, 22 and 40 after immunization, as well as untreated mice, showed only a few DEC-205+ cells with dendritic morphology in the outer skin layers (data not shown).

Despite the persistence of a visible depot of injected material in the s.c. tissue, it is hard to know how much immunogenic peptide is available at different days after immunization. However, the above observations indicate that both macrophage infiltration and dendritic cell recruitment to the s.c. site of immunization are only temporary after peptide and adjuvant injection, and suggest that remission correlates with diminished effective PLP 139–151 peptide presentation to autoreactive T cells in the draining lymph nodes. Indeed, the frequency of PLP 139–151 peptide-specific IFN-{gamma}-producing T cells and the amount of CD4+ and CD11b+ cells in the CNS infiltrate decreased slowly during spontaneous recovery, following the observed changes in the immunization site.


    Concluding remarks
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 
We suggest that in PLP 139–151 peptide-induced EAE the consumption of exogenous antigen and/or the lack of a long-lasting adjuvant effect lead to reduced inflammation and antigen-presenting cell recruitment and activation in the immunization site, and ultimately to diminished migration of antigen-loaded activated dendritic cells to the draining lymph nodes. Thus, autoreactive T cells would be stimulated only for a short interval in the draining lymph nodes and this would lead to a self-limited disease. Our hypothesis is in agreement with data showing that after s.c. administration of protein in adjuvant, the ability of lymph node dendritic cells to stimulate ex vivo antigen-specific hybridoma T cells peaked at day 3 and 6 after immunization, being negligible already at day 10 (26). The possibility that CNS inflammation is self-limited and EAE spontaneously resolves due to the lack of sustained stimulation of autoreactive T cells in the peripheral lymphoid organs is also supported by the finding that a second injection with PLP 139–151 peptide plus adjuvant in SJL mice which have recovered from active EAE induces a relapse (27).

Chronic relapsing-remitting EAE has been induced either with different protocols from ours (injection of different doses of PLP 139–151 peptide plus adjuvant without treatment with pertussis toxin, immunization with whole CNS homogenate, passive transfer of activated autoreactive T cells) or in mouse strains other than SJL (2831). In both cases, chronic EAE might have a long duration because of the stimulation of multiple waves of autoreactive T cells over time. One possibility is that immunogenic peptides are available for a long time at the immunization site and the local inflammation is sustained, as suggested by the finding that amputation of the hind feet to remove antigenic depots prevented subsequent episodes of clinical disease in a guinea pig chronic-relapsing EAE model (32). A second possibility is that an abundant release of self-antigen from the damaged CNS tissue leads to sustained activation of already primed T cells and/or recruitment of autoreactive T cells with new specificities (33). A third hypothesis suggests that, in autoimmune disease target organs, chronic inflammation can drive the formation of intraparenchymal organized lymphoid structures, where autoreactive T cell re-stimulation takes place (34). The three above mentioned possibilities are not mutually exclusive and can all contribute to the establishment of chronic autoimmune diseases. For instance, the persistence of the exogenous stimulus into the immunization site may both sustain local inflammation and maintain autoreactive T cell stimulation for sufficient time to allow chronic damage of the target organ and establishment of the other mechanisms of chronic autoimmunity.

A further point of discussion concerns our evidence that regulatory T cells do not determine spontaneous remission of acute EAE (here and 10). We suggest that the contribution of T cell regulatory networks to acute immune responses is negligible, whereas they are established and play a major role in chronic autoimmunity.

In conclusion, autoimmune diseases have an acute clinical course and do not self-maintain, unless there is either a major damage of the target organ, which can lead to autoreactive T cell re-stimulation by endogenous self-antigen, or a persistence of the inducing stimulus. Our hypothesis has important implications for the understanding of chronic autoimmune diseases, which we suggest are always maintained by repeated stimulation of autoreactive T cells. Indeed, it is tempting to hypothesize that infectious agents which have been involved in triggering human autoimmune diseases may play a major role in their chronicization, when the pathogens are able to persist in the host (3537). A persisting infectious agent may contribute to the establishment of chronic autoimmunity via the following mechanisms: (i) it can provide cross-reactive antigens, (ii) it can damage host cells or influence their protein synthesis, leading to unveiling of cryptic epitopes (38), (iii) it can induce chronic inflammation, allowing stimulation of multiple waves of autoreactive T cells, and (iv) it can sustain autoreactive T cell stimulation until the target organ is damaged enough that late mechanisms of autoimmune response amplification (i.e. epitope spreading, formation of lymphoid tissue islets) can take place.


    Acknowledgments
 
We thank Dr Rodolfo Lorenzini and his co-workers for their help at the animal facility of Istituto Superiore di Sanità. This work was supported by Ministero della Sanità-Istituto Superiore di Sanità (I and II Progetto Sclerosi Multipla), Progetto Associazione Italiana Sclerosi Multipla 1997–2000 (AISM), Progetto Finalizzato Consiglio Nazionale delle Ricerche `Biotecnologie', European Community Contract no. BMH4-CT98-3703 and Ministero dell'Università e della Ricerca Scientifica e Tecnologica 40%.


    Abbreviations
 
CFA complete Freund's adjuvant
CNS central nervous system
COX cyclooxygenase
EAE experimental allergic encephalomyelitis
MS multiple sclerosis
PG prostaglandins
PLP proteolipid protein

    Notes
 
5 Present address: Laboratorio di Fisiopatologia, Centro Ricerca Sperimentale, Istituto Regina Elena, via delle Messi d'Oro 156, Rome 00158, Italy Back

6 Present address: Istituto Nazionale per le Malattie Infettive IRCSS `La Sapienza' CRAIDS, via Portiense 292, Rome 00149, Italy Back

Transmitting editor: G. Doria

Received 8 October 1999, accepted 3 February 2000.


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 Abstract
 Introduction
 Methods
 Results and discussion
 Concluding remarks
 References
 

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