Citrullinated myelin basic protein induces experimental autoimmune encephalomyelitis in Lewis rats through a diverse T cell repertoire

Journal of Neuroimmunology 88 Ž1998. 21–29

Citrullinated myelin basic protein induces experimental autoimmune encephalomyelitis in Lewis rats through a diverse T cell repertoire Ligong Cao b

a,b,)

, Deming Sun

a,b

, John N. Whitaker

a,b,c

a Department of Neurology, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294-0007, USA Center for Neuroimmunology, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294-0007, USA c Neurology and Research SerÕices of the Birmingham Veterans Medical Center, Birmingham, AL, USA

Received 29 October 1997; revised 26 February 1998; accepted 26 February 1998

Abstract An increased proportion of citrullinated MBP ŽMBP-C8. occurs in the brains of multiple sclerosis ŽMS. patients. In this study, MBP-C8 from guinea pig ŽGP. brains was isolated and found encephalitogenic in Lewis rats upon immunization. An encephalitogenic T cell line selected with MBP-C8 preferentially reacted with MBP-C8 over unmodified MBP. This T cell line responded weakly to the dominant encephalitogenic epitope, GP-MBP peptide 70–88, and did not display restricted TCR b-chain usage Žsuch as Vb 8.2.. The distinctive features of MBP-C8 were also demonstrated by its ability to reinduce active EAE in 70% of rats which had recovered from unmodified MBP induced EAE. These findings raise the possibility that citrullinated MBP may elicit a different pathogenic T cell repertoire for the recurrent phases of inflammatory demyelination. q 1998 Elsevier Science B.V. All rights reserved. Keywords: T cells; Experimental autoimmune encephalomyelitis; MBP isomer; Citrulline; Multiple sclerosis

1. Introduction Multiple sclerosis ŽMS. is an inflammatory demyelinating disease affecting the central nervous system ŽCNS. myelin of humans. It typically causes recurrent acute phases and more chronic, progressive neurologic disability ŽMcFarlin and McFarland, 1982.. An autoimmune immunopathogenesis is strongly suspected, but the antigenŽs. targeted and the mechanisms involved are unclear. Because of its strong encephalitogenic property, myelin basic protein ŽMBP. has long been a candidate antigen for MS. MBP accounts for approximately 35% of CNS myelin proteins and, as a consequence of alternate splicing of the seven exons of the single MBP gene, exists in four ŽRoth et al., 1987. or more isoforms. In addition to these isoforms, which vary in relative amounts during myelinogenesis and remyelination ŽTakahashi et al., 1985; Boylan et al., 1990., the number of different MBP molecular species and epitopes is further expanded by the creation of charge isomers resulting from post-translational modifications such as phosphorylation ŽDeibler et al., 1975., deamidation )

Corresponding author. Tel.: q1 205 9757266; fax: q1 205 9757667.

0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 0 6 3 - 0

ŽChou et al., 1976. and citrullination ŽWood and Moscarello, 1989.. These isomers can be separated by cation exchange chromatography under strong alkaline conditions during which the least modified and most cationic isomer, designated as MBP-C1, elutes last and the least cationic isomer and most citrullinated isomer, designated as MBP-C8, passes through the column unretarded ŽWood and Moscarello, 1989.. In MBP-C8 protein, six of the 19 arginyl residues are selectively citrullinated resulting in a loss of six positive charges ŽWood and Moscarello, 1989.. MBP-C8 is the dominant isomer in early myelinogenesis with a ratio of C8rC1 at 7.5 and this ratio becomes as low as 0.82 in the normal adult ŽMoscarello et al., 1994.. MBP-C8 is relatively increased in post-mortem brain tissue from individuals with MS ŽMoscarello et al., 1994.. Especially, this was noted in one case of the fulminating, or Marburg, form of the disease in which the C8rC1 ratio rose to 6.7 with 17 of the 19 arginyl residues of MBP converted to citrulline ŽWood et al., 1996.. Our previous investigations ŽZhou et al., 1993, 1995. have demonstrated that citrullination of MBP and the varied length of acylation in the aminoterminus of MBP affects the immune response to MBP.

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MBP-C8 reactive T cells have been found in normal and MS bloods ŽMartin et al., 1994.. The present study addresses whether MBP-C8 induces EAE in Lewis rats, whether it elicits distinct pathogenic T cell subsets, and whether it does so by expressing unique encephalitogenic epitopeŽs.. We show that, in comparison to uncitrullinated MBP, the presence of citrulline residues in MBP changes its T cell epitopes and the repertoire of encephalitogenic T cells in the Lewis rat.

2. Materials and methods 2.1. Animals Female Lewis rats of seven to eight weeks of age, purchased from Charles River Laboratories ŽRaleigh, NC., were used in all the experiments. 2.2. Antigens MBP was extracted from GP or Rat ŽRt. brain tissue ŽPelfreez, Rogers, AR. or from post-mortem human brain. Delipidation, acidic extraction and isolation of charge isomers of MBP by cation exchange chromatography were according to previously described methods ŽWhitaker and Seyer, 1979.. By cation exchange chromatography at pH 10.5 in the presence of urea, MBP-C8 is unretained and MBP-C1 is eluted as the last peak with a NaCl gradient ŽWhitaker and Seyer, 1979; Wood and Moscarello, 1989.. MBP-C8 was further purified by high performance liquid chromatography ŽHPLC. with the fractions containing MBP-C8 identified by immunochemical testing ŽZhou et al., 1993.. GP MBP peptide 70–88 was synthesized in the UAB Cancer Center core facility, and MBP peptide 86–98 was purchased from Peninsula Laboratories ŽSan Carlos, CA. ŽTable 1.. Other peptides, including altered peptides derived from the sequence of GP MBP peptide 70–88 ŽTable 1., were synthesized in the UAB Cancer Center

Table 1 The sequences of MBP peptides Peptides

Sequences

Ac1–9 Ac1–21 GP MBP15–30 GP MBP21–40 GP MBP51–70 GP MBP70–88 ŽA76. 69–85a ŽP80. 69–87 a GP MBP86–98 GP MBP98–117

Ac–ASQKRPSQR Ac–ASQKRPSQRHGSKYLATASTM LATASTMDHARHGFLP MDHARHGFLPRHRDTGILDS APKRGSGKDSHHAARTTHYG GSLPQKSQRSQDENPWHF YGSLPQKAQRSQDENPV b YGSLPQKSQRPQDENPWH b VHFFKNIVTPRTP PPPSQGKGRGLSLSRFSWGA

a

The letter number in parenthesis indicates amino acid substituted at that residue. b Bold letters represent the amino acid substitution.

Peptide Synthesis Laboratory ŽSchlagel et al., 1997. or at St. Jude Children’s Research Hospital, Memphis, TN ŽSun et al., 1995.. 2.3. Characterization of GP MBP isomers The GP MBP isomers eluted from the HPLC columns were immunologically characterized using MBP-specific monoclonal antibodies ŽmAbs. including mAb 845D3 anti-human MBP peptide 80–89 ŽPrice et al., 1986., mAb F28C4 anti-human MBP peptide acetyl 1–9 ŽZhou and Whitaker, 1992. and mAb F32F9 anti-human peptide 10– 19 ŽZhou et al., 1993.. By enzyme-linked immunoassay ŽELISA. ŽSchlagel et al., 1997., the first two mAbs bind to both MBP-C8 and MBP-C1, but mAb F32F9 binds only to MBP-C1. Briefly, 96-well microtiter plates were coated with MBP or column fractions at 0.4 m g welly1 . After blocking with 1% BSA, 75 m l of mAb cell culture supernatants were added to the wells of the plates, followed by incubation with 1:500 diluted second Ab which was goat anti-mouse IgŽH q L.-AP ŽSouthern Biotechnology, Birmingham, AL.. The plates were developed by substrate solution of pNPP tablets ŽSigma. and read at 405 nm 10 min later. The GP MBP-C8 was also characterized by Western blotting in which the protein was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. This was followed by serial incubation with mAb 845D3 and alkaline phosphatase labelled goat anti-mouse Ig ŽSouthern Biotechnology, Birmingham, AL.. Bands on the membrane were then visualized with alkaline phosphatase substrate solution. 2.4. ActiÕe EAE induction Active EAE was induced in Lewis rats by the subcutaneous Žs.c.. injection of GP MBP-C1 or GP MBP-C8 Ž50 m g raty1 . emulsified in complete Freund adjuvant ŽCFA.. Reinduction of active EAE was attempted by repeat s.c. inoculation of GP MBP-C1 or GP MBP-C8 Ž200 m g raty1 . emulsified in CFA. The severity of EAE was scored according to the following clinical scale: 0, no clinical signs; 1 q , mild tail weakness; 2 q , complete loss of tail movement andror hindlimb paresis; 3 q , moderate hindlimb paralysis; 4 q , total hindlimb paralysis; 5 q , moribund or death. 2.5. Establishment of MBP-specific T cell lines from Lewis rats Lewis rats were immunized with GP MBP-C8 in CFA. The draining lymph nodes were removed 14 days later which was at the height of disease. A single cell suspension was made and placed in stimulation medium made of RPMI-1640 containing antibiotics, 1 mM L-glutamine, 5 = 10y5 M 2-ME, and 10% heat inactivated fetal calf serum ŽFCS.. 2 = 10 7 lymph node cells were stimulated with 250

L. Cao et al.r Journal of Neuroimmunology 88 (1998) 21–29

m g of GP MBP-C8 in 10 ml of stimulation medium for three days. The activated T cells were isolated by Ficoll density gradient centrifugation, washed with RPMI-1640 twice and further cultured in growth medium containing 10% heat inactivated horse serum, antibiotics, L-glutamine, and 2-ME as well as 5% ConA-stimulated spleen cell supernatant for the source of IL-2. Four days later, the resting T cells were restimulated with GP MBP-C8 in the presence of irradiated thymocytes from naive Lewis rats as antigen present cells ŽAPCs. in the stimulation medium using 1% normal rat serum as a substitute of FCS, followed by the culture in IL-2 containing growth medium. The 7-day-cycle was repeated several times. The GP MBPC8 specific T cell line was designated RT-C8. The GP MBP-C1 specific T cell line, designated RT-C1, was developed as above, except that the GP MBP-C1, rather than GP MBP-C8, was used for immunization and T cell stimulation. A T cell line, specific for GP MBP peptide 70–88 designated LR88L1 ŽZhou et al., 1994., was developed by immunizing with unmodified GP MBP-C1 and selecting with GP MBP peptide 70–88. 2.6. Induction of adoptiÕe transfer EAE To adoptively transfer EAE, MBP-C8 specific T cells ŽRT-C8. were stimulated with GP MBP-C8 Ž20 m g mly1 . for three days in the presence of irradiated syngeneic thymocytes as APCs. Activated T cells were isolated by Ficoll density gradient centrifugation and washed with Hank’s solution. Each recipient rat intraperitoneally Ži.p.. received 0.5–1.5 = 10 7 T cells suspended in PBS. The scoring of disease severity followed the criteria used for active EAE. 2.7. Serological response of MBP isomers in Lewis rats

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triplicate wells of flat-bottom 96-well microtiter plates in the presence of antigens at a concentration of 10 m g mly1 or otherwise as indicated. The plates were incubated under 5% CO 2 and 378C for 72 h. During the last 18 h of culture, the cells were pulsed with w3 Hxthymidine at 0.5 m Ci welly1 after cells were harvested and w3 Hxthymidine incorporation measured. The T cell proliferation assay of lymph node cells was performed as above, except that the number of cells seeded in the 96-well plates was 4 = 10 5 cells welly1 . 2.10. FACS analysis for phenotyping of T cell lines Cell surfaces were stained indirectly and analyzed by FACS. Viable resting T cells were isolated by Ficoll gradient centrifugation. After washing with RPMI-1640, the T cells Ž2 = 10 5 . were incubated with mouse mAbs including R73 Žanti-TCR-ab ., R78 Žanti-TCR Vb 8.2., OX35 Žanti-CD4., anti-Vb 10 and anti-Vb 16 ŽPharMingen., followed by the second Ab FITC labelled goat anti-mouse Ig ŽPharMingen. before analysis by FACStar ŽBecton Dickinson, Mountain View, CA.. 2.11. Analysis of TCR Vb by polymerase chain reaction (PCR) A reverse transcriptase polymerase chain reaction ŽRT– PCR. was performed following the protocol previously described ŽSun et al., 1994.. Briefly, T cells were stimulated with specific antigen and the total RNA was extracted three days later. The contamination of TCR mRNA from APCs has been greatly decreased by choosing a TCR-MHC-IIq APC line LOA ŽSun et al., 1994.. cDNA was synthesized from the total RNA of T cells using a reverse-transcriptase cocktail containing 0.5 m M random primer ŽPharmacia., 500 m M dNTP, and 200 units Moloney murine leukemia virus reverse transcriptase ŽGibco. in

Sera collected from the rats six to seven weeks after immunization with MBP-C1 or C8 were tested by ELISA to detect humoral immunoresponse to both MBP isomers. The MBP-plated microtiter plates were incubated with a 1:500 dilution of serum followed by a 1:500 dilution of goat anti-rat IgŽH q L.-AP as the second antibody. 2.8. Histopathology The lumbosacral part of rat spinal cords was fixed in 10% formalin and embedded in paraffin. Sections Ž8 m m. of duplicate longitudinal and transverse sections were stained with hematoxylin and eosin ŽH–E. for evaluation of inflammatory infiltration. 2.9. T cell proliferation assay For the proliferation assay of T cell lines, 2.5 = 10 4 resting specific T cells were co-cultured with 2 = 10 6 irradiated Ž2500 Rads. syngeneic thymocytes in each of

Fig. 1. ELISA demonstrating the reaction of MBP peptides specific mAbs with human and guinea pig MBP isomers. 96-well microtiter plates were coated with the indicated proteins at 0.4 m g welly1 and blocked with 1% BSA in PBS. The neat hybridoma cell culture supernatants Ž75 m l., as indicated in the insert, were added to each well, followed by incubation with a 1:500 dilution of goat anti-mouse IgŽHqL.-AP. The plates were finally developed by substrate pNPP solution for 10 min and read at 405 nm. The pattern of mAb reactivity with MBP-C1 and MBP-C8 is the same for human and GP.

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L. Cao et al.r Journal of Neuroimmunology 88 (1998) 21–29

Fig. 2. ELISA for serological response of Lewis rats. Human and GP MBP isomers and GP MBP peptide 70–88, as indicated in insert, were plated at 0.4 m g mly1 after which was added 75 m l of a 1:500 dilution of sera obtained from rats recovered Žsix to seven weeks after immunization. from EAE induced with either GP MBP-C1 Ž ns 4. or GP MBP-C8 Ž ns 4.. An amount of 100 m l goat anti-rat IgŽHqL.-AP, diluted 1:500, was added to each well, followed by visualization with pNPP solution and read at 405 nm. The serum antibodies, controlled by normal serum, react well with MBP isomers from either of the two species.

Fig. 4. T cell proliferation assay for analysis of T cell response to MBP isomers and encephalitogenic peptide. T cell proliferation assay was performed in 96-well flat bottom microtiter plates. T cells Ž2.5=10 4 cells welly1 . of the LR88L1 line, generated by GP MBP peptide 70–88 ŽA., or of the RT-C8 line, selected by GP MBP-C8 ŽB. were stimulated with the indicated antigens Ž25 m g mly1 . plus irradiated syngeneic thymocytes Ž2=10 6 cells welly1 . as APCs for 72 h. During the last 18 h of culture, cells were pulsed with w3 Hxthymidine at 0.5 m Ci welly1 before the cell harvest. Similar to results with lymph node cells Žsee Fig. 3. but even more selective, a preferential response is noted to the different isomers.

PCR products were electrophoresed on 1.5% agarose gel stained with ethidium bromide. reverse transcriptase buffer at 428C for 42 min, followed by heat inactivation and cooling for 3 min on ice. The synthesized cDNA was subjected to PCR in 40 m l of a solution containing 10 = amplification buffer, dNTPs, 0.5 unit Taq polymerase ŽPerkin–Elmer., 0.2 m M TCR Vbspecific primer and 0.5 m M TCR C b-specific primer ŽSun et al., 1994.. The reaction was carried out in a Perkin– Elmer thermocycler for 35 cycles with 948C for 1 min, 568C for 45 s and 728C for 45 s in each cycle. The final

Fig. 3. T cell proliferation assay of lymph node cells from Lewis rats immunized with GP MBP isomers. The proliferation assay was performed in 96-well flat bottom microtiter plates. Lymph node cells Ž4=10 5 cells welly1 . from the Lewis rats with EAE induced by GP MBP-C1 or GP MBP-C8 at peak disease were stimulated with indicated antigens Ž25 m g mly1 . for 72 h and pulsed with w3 Hx thymidine Ž0.5 m Ci welly1 . for 18 h prior to cell harvest. A difference in T cell reactivity to the different isomers is noted.

3. Results 3.1. Immunochemical difference between GP MBP-C1 and MBP-C8 We have previously shown that human MBP-C8 reacted with mAb 845D3 and F28C4 but not F32F9, whereas all

Fig. 5. T cell proliferation assay of RT-C8 and RT-C1 T cell lines with different concentrations of antigens. The specific T cells of RT-C1 Žright panel., elicited by GP MBP-C1, and RT-C8 Žleft panel., selected by GP MBP-C8, were seeded in 96-well plates at 2.5=10 4 cells welly1 and stimulated with 0–10 m g mly1 of GP MBP isomers or GP MBP peptide 70–88 as indicated in insert, in the presence of irradiated syngeneic thymocytes Ž2=10 6 cells welly1 .. Cells and antigens were incubated for 72 h during the last 18 h of which cells were pulsed with w3 Hxthymidine Ž0.5 m Ci welly1 . before cell harvest. The vertical bars represent the standard errors. The differences in reactivities of T cells lines directed at different GP MBP isomers are evident. Welch t-test was applied for statistical analysis of the T cell line proliferation result. RT-C1 responds to MBP-C1 significantly better than to MBP-C8, while RT-C8 preferentially reacts to MBP-C8 over MBP-C1 Ž0.02 ) P ) 0.002..

L. Cao et al.r Journal of Neuroimmunology 88 (1998) 21–29

Fig. 6. T cell proliferation assay of RT-C1 and RT-C8 T cell lines with a panel of MBPs and peptides. The specific T cells of RT-C1 and RT-C8 line were placed in 96-well plates Ž2.5=10 4 cells welly1 . and stimulated by a panel of MBP and MBP peptides Ž10 m g mly1 . Žsee Table 1. in the presence of irradiated syngeneic thymocytes for 48 h. During the last 12 h of culture, cells were pulsed with 0.5 m Ci welly1 before cell harvest. Note the prominent reactivity of RT-C1 to GP MBP and peptide 70–88 and very weak reactivity by RT-C8 to this peptide and other altered peptides.

the three antibodies react with the human non-citrullinated MBP-C1 ŽZhou et al., 1993.. The same binding pattern with these mAbs by ELISA was noted for the MBP-C8 and MBP-C1 prepared from GP brains ŽFig. 1. as well as the Western blotting Ždata not shown.. The molecular weight of GP MBP-C8 is 18.5 kd as determined by SDS-PAGE and RP-HPLC showed common retention time for MBP-C1 and MBP-C8, either from GP or human Ždata not shown.. 3.2. Immune response to MBP-C8 in Lewis rats The humoral response to GP MBP-C1 and GP MBP-C8 was compared. Immune sera, collected six to seven weeks

25

from Lewis rats after immunization with GP MBP-C8 or MBP-C1, bound to both molecules as well as to the human MBP-C1 and MBP-C8 ŽFig. 2.. GP MBP peptide 70–88, the major encephalitogenic peptide for the Lewis rat, was also recognized by either ŽFig. 2.. In contrast to this shared humoral reactivity to both MBP isomers, lymph node cells from rats immunized with either GP MBP-C1 or GP MBP-C8 preferentially responded to the isomer used as immunogen ŽFig. 3.. Such a preferential response to GP MBP-C8 was also observed with T cell lines RT-C8, selected by GP MBP-C8 ŽFig. 4B.. Although the response of RT-C8 to human MBP-C8 was much less than to either GP MBP isomer ŽFig. 4B., human MBP-C8 effected a stimulation 10-fold Žstimulation index of 12 compared to 1.2. greater than human MBP-C1. The peptide 70–88-generated T cell line LR88L1 responded well to both GP MBP-C1 and GP MBP-C8 and only marginally, but equally, to human MBP-C1 and MBP-C8 ŽFig. 4A.. 3.3. T cells specific for GP MBP-C8 do not dominantly respond to GP MBP peptide 70–88 In order to characterize and compare antigenic specificity of RT-C8 cells with MBP-reactive T cells elicited by isomer MBP-C1, RT-C1 line was established from animals immunized with GP MBP-C1. Both RT-C8 and RT-C1 were then assayed against varying concentrations of GP MBP isomers and GP MBP peptide 70–88. Response to a single optimal concentration of 25 m g mly1 ŽFig. 4B. and over a range of antigen concentrations ranging from 0.08 to 10 m g mly1 revealed similar results ŽFig. 5.. RT-C8 cells reacted well with GP MBP-C8, slightly less well with GP MBP-C1 and poorly to GP MBP peptide 70–88 ŽFig. 5.. In contrast, RT-C1 cells responded strongly to GP MBP-C1 and GP MBP peptide 70–88, but weakly to GP MBP-C8. To further compare the fine specificities of these two T cell lines, their responses to a panel of MBP peptides with or without amino acid alterations were examined. There were striking differences between the RT-C1

Fig. 7. Induction of adoptiÕe passiÕe EAE in Lewis rats by T cell line RT-C8 responsiÕe to GP MBP-C8. After four cycles of stimulation with GP MBP-C8 and irradiated rat thymocytes as APCs, RT-C8 T cells were injected i.p. into eight week-old Lewis rats. The injected cell number was 0.5 = 10 7 cellsreach Žblank diamond. for three rats and 1.5 = 10 7 cellsreach Žfull diamond. for two rats. The clinical severity was scored as following: 0, no clinical signs; 1 q , mild tail weakness; 2 q , complete loss of tail movement andror hindlimb paresis; 3 q , moderate hindlimb paralysis; 4 q , total hindlimb paralysis; 5 q , moribund or death.

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26 Table 2 The phenotypes of MBP specific T cell lines

3.5. GP MBP-C8-specific T cells are encephalitogenic

Phenotypesa

RT-C1

RT-C8

TCR-ab TCR Vb 8.2 TCR Vb 10 TCR Vb 16 CD4

98.2 b 60.8 3.9 2.2 92.5

92.4 7.4 0.5 5.6 91

a

Indirect immunofluorescence was used to stain the cell surfaces of the T cell lines as detailed in Section 2. b The numbers represent the percentages of positive cells.

and RT-C8 T cell lines ŽFig. 6. in their responses to GP MBP peptide 70–88 region and its altered peptides. Of the peptides tested, RT-C8 cells showed no specific response but did proliferate weakly to GP MBP peptide 70–88. The pattern of reactivity of RT-C1 cells to the dominant epitope of GP MBP peptide 70–88 or related peptides was as expected except for the detection of a response to GP MBP peptide 51–70. 3.4. GP MBP-C8 is highly encephalitogenic in Lewis rats Nine Lewis rats at seven weeks old in three groups Ž3 ratsreach. received a s.c. immunization with three different doses of GP MBP-C8 Ž25, 100 or 200 m g raty1 . respectively in CFA for active EAE induction. As low as 25 m g raty1 of GP MBP-C8 induced severe EAE. The clinical course was monophasic with onset at 11–12 days and peak disease Žmost suffering paraplegia. at 14–17 days after immunization. Animals started to recover by days 18–19. Most of rats completely recovered from acute EAE induced by MBP-C8 as well as MBP-C1, although some rats did show a mild disease for an extended period of time and very few rats showed clinical fluctuation during recovering phase. No differences in clinical course was noted among the varied doses of the immunogen. Histopathological examination of spinal cords from the acute disease phase displayed extensive inflammatory infiltration without noticeable demyelination Ždata not shown..

The RT-C8 T cells, which preferentially reacted with GP MBP-C8 over GP MBP-C1 and GP MBP peptide 70–88 ŽFig. 4BFigs. 5 and 6., were also encephalitogenic. RT-C8 T cells were activated for 3 days with 20 m g mly1 of GP MBP-C8. Naive Lewis rats receiving 0.5 = 10 7 or 1.5 = 10 7 cells clearly expressed clinical disease by day 5. Overt signs, notably paraplegia, resolved over about days 6 or 7 ŽFig. 7.. Histopathological examination during the acute phase of disease revealed extensive inflammatory infiltration in the lumbar spinal cord Ždata not shown.. 3.6. DiÕersity of TCR V gene usage by RT-C1 and RT-C8 T cells RT-C1 and RT-C8 cells were phenotyped by a panel of available mAbs to cell surface molecules ŽTable 2.. The RT-C1 cells were 98.2% positive for TCR-ab , and 60.8% positive for Vb 8.2 gene expression. RT-C8 cells were 92.4% positive for TCR-ab , but only 7.4% were positive for Vb 8.2. To further determine the TCR Vb-chain usage of RT-C8 T cell line at mRNA level, a panel of 22 primers specific to rat TCR Vb segments and a C b-specific oligonucleotide were applied in a RT–PCR. Although this RT–PCR is not aimed to quantitate precisely the TCR Vb gene expression, it showed that the TCR b-chain usage of RT-C8 line is quite heterogeneous ŽFig. 8. and not restricted to Vb 8.2, which is consistent with the findings of cell surface staining. These results indicate that MBP-C8 stimulates a different T cell repertoire. 3.7. Animals recoÕered from GP MBP-C1-induced EAE are not resistant to reinduction of EAE by GP MBP-C8 Active EAE in Lewis rats is typically acute, monophasic and refractory to subsequent reinduction with GP MBP ŽMacPhee and Mason, 1990.. To further characterize differences of the encephalitogenic epitopes of GP MBP-C1 and MBP-C8 and their ability to cross-tolerize, animals recovered from MBP-C1-induced EAE were tested for

Fig. 8. PCR analysis of TCR b-chain usage of MBP-C8-specific T cell line RT-C8. The total cytoplasmic mRNA was isolated from T cell line RT-C8 three days after stimulation with GP MBP-C8 plus irradiated LOA and converted to cDNA. The cDNA was amplified with PCR in a 40-m l volume containing 0.5 m M TCR C b oligonucleotide primer, 0.2 m M TCR Vb-specific oligonucleotide primer, 0.2 mM dNTP and 0.5 unit Taq DNA polymerase. The reaction was carried out in a thermocycler with 35 amplification cycles of 1 min at 948C, 45 s at 568C and 45 s at 728C. The PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide.

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Table 3 Reinduction of active EAE in the Lewis rats Primary EAE induction

Reinduction of EAE

Group

Immunogen

Incidence

Clinical severity

Immunogen

Incidence

Increased clinical severity a

CNS inflammationb

1 2 3 4

C1rCFA C1rCFA C8rCFA CFA

4r4 7r7 9r9 0r4

3.5 3.9 3.3 0

C1rCFA C8rCFA C8rCFA C8rCFA

0r4 5r7 0r9 4r4

0 1.2 0 4

q qq q qq

a

Increased clinical seÕerity was calculated by the division of accumulation of increased clinical scorerthe number of rat showing the reappearance of neurological signs. b CNS inflammation was graded as: y, no inflammation: q, rare parenchyma infiltration; qq, significant meningitis andror inflammatory infiltration in parenchyma.

EAE reinduction with either GP MBP-C1 or GP MBP-C8 in CFA. EAE was reinduced with GP MBP-C8 in five of seven such rats previously recovered from EAE induced by GP MBP-C1 ŽTable 3, Group 2.. Following the immunization with MBP-C8, the neurological signs appeared from 1–4 weeks later and remained for a varying length of time from several days to months. Unlike the typical ascending paralysis in acute EAE, the recurrent EAE showed more scattered neurological deficits, such as hindlimb paralysis with or without tail weakness, or asymmetrical hindlimb weakness. The pathological examination of spinal cords from animals with MBP-C8 reinduced EAE, one to two months after second immunization and when the recurrent signs were stable or resolved, revealed chronic perivascular infiltration ŽFig. 9.. The clinical recurrence of EAE was not found in animals whose first and second immunizations were with the same GP MBP iso-

Fig. 9. The histopathological examination of the spinal cords from the Lewis rats with EAE. The 8 m m sections of the paraffin embedded lumbosacral part of spinal cord from rats with reinduced EAE by MBP-C8 Žfrom Group 2, Table 3. were stained with hematoxylin–eosin. A meningeal and perivascular inflammatory infiltration can be seen. ŽA. =100, ŽB. =400.

mers ŽTable 3, Groups 1 and 3.. Pretreatment with CFA supplemented with Mycobacterium ŽH37Ra. in four Lewis rats ŽTable 3, Group 4. failed to protect the animals from subsequent induction of active EAE with GP MBP-C8 in CFA.

4. Discussion On the basis of its isoforms and isomers, MBP is actually a family of molecules with multiple members ŽMoscarello et al., 1994.. MBP-C8, the citrullinated isomer, has lessened cationic charge and a lessened ability to form acidic lipid vesicles ŽWood and Moscarello, 1989.. Its early appearance in myelinogenesis ŽMoscarello et al., 1994., distribution in the intraperiod line of compact CNS myelin ŽMcLaurin et al., 1993. and relative increase in CNS myelin from MS brain tissues ŽMoscarello et al., 1994. indicate that the citrullination of MBP leads to distinct, if not unique, properties and roles among the array of MBP molecules. The excessive citrullination in the MBP from the post-mortem brain tissue of a patient dying of fulminating MS ŽWood et al., 1996. prompted our speculation ŽWhitaker and Mitchell, 1996. on the autoimmunogenicity created by the citrullination of human MBP. The questions remain whether the autoreactive T cells elicited by distinct MBP isomers have unique cellular and molecular identities and whether they are implicated in the pathogenesis of the anti-myelin autoimmune disease. To address these questions, we successfully isolated MBP-C8 from guinea pig brains and generated T cells specific to GP MBP-C8 in Lewis rats. Remarkably, we found that GP MBP-C8, like its unmodified counterpart, MBP-C1, is highly encephalitogenic in Lewis rats upon active immunization and that adoptive transfer of MBPC8-specific T cells leads to severe clinical EAE in these animals. We have further demonstrated that GP MBP-C8 is capable of re-inducing EAE in Lewis rats that had recovered from MBP-C1-induced EAE. This EAE reinduction by GP MBP-C8 following earlier exposure to MBP-C1 also resulted in a shift in clinical features and chronic CNS inflammatory infiltration as well as, in some rats, patchy

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CNS myelin faint staining by Luxol fast blue which indicates the presence of demyelination Ždata not shown.. Based on the previous reports that EAE-pathogenic T cells not only cause disease but also induce activation of EAEregulatory T cells that act by preventing animals from re-induction of disease ŽEllerman et al., 1988; Lider et al., 1988; Sun et al., 1988., we hypothesized that MBP-C8 might have reinduced EAE by priming distinct pathogenic T cell subsets which might escape the control of regulatory T cells specific for MBP-C1-primed T cells and cause inflammatory infiltration in CNS. Secondly, the impact of citrullination of MBP on T cell response was examined. It was found that the lymph node cells from the Lewis rats immunized with GP MBP-C8 preferentially proliferated in culture with GP MBP-C8 over GP MBP-C1 and this preferential response persists in GP MBP-C8-generated T cell line RT-C8. Although this T cell line did not react to human MBP-C1, it still responded appreciably to human MBP-C8, implying that citrullination enhanced T cell recognition of the human MBP-C8 molecules. Since the difference on T cell response to the citrullinated and non-citrullinated MBP isomers has been shown in our study, we further wondered if the immunodominant epitope of MBP shifts upon the citrullination. GP MBP peptide 70–88 is the dominant T cell epitope of MBP in Lewis rats ŽKibler et al., 1977. with a subdominant epitope located in peptide 86–98 ŽOffner et al., 1989.. In our experiments, it was interesting to find that the dominant T cell epitopes of GP MBP-C8 have become obscure, since MBP-C8-generated RT-C8 T cell line responded weakly to MBP peptides 70–88 and 86–98 as compared to their strong response to intact GP MBP-C8. Based on the sequence information of human MBP-C8 ŽWood and Moscarello, 1989., neither peptide 70–88 nor 86–98 from GP MBP is expected to be citrullinated. In fact, a peptide 70–88-generated T cell line LR88L1 did strongly respond to GP MBP-C8, implying no modification in residues 70–88 of this protein. Thus, a modification of GP MBP-C8 peptide 70–88 is not the explanation for a shift of the dominant epitope of GP MBP-C8 in Lewis rats from MBP peptide 70–88. The possible mechanisms by which the immunodominant epitopes of MBP shift upon citrullination are as follows: Ž1. The conversion of arginine residue to citrulline accompanying the loss of positive charges may have an impact on the affinity of MBP fragments to MHC Class II molecules; Ž2. The change of primary as well as secondary molecular structure may endow the MBP and its fragments with resistance to the degradation by proteinases ŽTsubata and Takahashi, 1989. and, thereby, the pattern of antigen processing in endocytic compartments may be altered; Ž3. Since the MHC-II molecules can bind the long peptides ŽFridkis-Hareli et al., 1995. and let them be subsequently trimmed by exopeptidases ŽNelson et al., 1997., instead of complete proteolysis during antigen processing, the variant fragments bound to MHC-II

could yield different length of flanking sequences outside the core peptides, which are important for activation of T cells ŽMuller et al., 1996.. All the aforementioned possibilities may result in a shift in antigen presentation and play an important role in selecting T cells specific to certain novel epitopes. This hypothesis was supported by our unpublished experiment in which degradation of MBP-C8 and MBP-C1 by lysosomal hydrolase cathepsin D consistently produced two different groups of peptides as determined by HPLC and mass spectrometry. The influence of citrullination on antigen presentation may be proved and clarified by the analysis of naturally processed peptides eluted from MHC-II molecules. Since citrullination of MBP isomers is not all or nothing ŽWood and Moscarello, 1989., varying citrullination and other post-translational modifications might be involved in modifying protein degradation. The assumption that GP MBP-C8 may have elicited distinct subsetŽs. of pathogenic T cells has also been supported by our observations that MBP-C8-specific T cells are phenotypically and pathobiologically different from the encephalitogenic T cells elicited by MBP-C1. For example, this T cell repertoire did not preferentially utilize TCR Vb 8.2, the common TCR gene segments utilized by the encephalitogenic T cell subsets elicited by GP MBP peptide 70–88 in the Lewis rat ŽGold et al., 1991.. The TCR Vb gene usage of MBP-C8-specific T cell line as determined by RT–PCR exhibits broad heterogeneity since all 22 tested TCR Vb-encoding mRNAs were found in this T cell line. Since the RT–PCR used was not quantitative, the exact percentage of MBP-C8-specific T cells bearing diverse TCR Vb will be further analyzed once the antibodies specific to diverse TCR Vb-chains are available. In the present investigation, evidence is provided that the secondary structure of the GP MBP molecule is changed as a consequence of citrullination, a finding previously observed in human MBP ŽWhitaker et al., 1992; Zhou et al., 1993.. GP MBP-C8 has been found to lose affinity to mAb F32 which was generated by a non-modified MBP peptide 10–19 and recognizes MBP-C1. Since the residues 10–19 in MBP-C8 is not citrullinated ŽWood and Moscarello, 1989., its loss of binding to mAb F32 indicates certain alterations of secondary structure of this citrulline-containing isomer. In summary, MBP-C8, the citrullinated isomer of GP MBP, has potent EAE-inducing activity in the Lewis rats. It elicits pathogenic T cell subsets having a different MBP epitope specificity and TCR usage than those T cells generated with non-citrullinated MBP isomer, MBP-C1. Such distinct immunological properties of the C8 isomer lead to a more heterogeneous autoimmune response. These findings raise the possibility that the same molecular isoform of MBP, with the additional structural diversity from post-translational modifications, may provide a basis for epitope spreading and permit recurrent bouts of inflammation in CNS.

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Acknowledgements This work was supported by the Research Service of the Veterans Administration, National Institutes of Health Grant NS29719, and National Multiple Sclerosis Society Grant RG2667. We thank Mrs. Qiao Han, Mrs. Sheher Sun and Ms. Linda Brent for excellent technical and secretarial assistance.

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