Autoreactive IgG to intracellular proteins in sera of MS patients

Journal of Neuroimmunology 99 Ž1999. 72–81 www.elsevier.comrlocaterjneuroim

Autoreactive IgG to intracellular proteins in sera of MS patients Fengmin Lu, Bernadette Kalman

)

Center for NeuroÕirology, MCP — Hahnemann UniÕersity, 245. N. 15th Street, Philadelphia, PA 19102-1192, USA Received 19 February 1999; received in revised form 19 May 1999; accepted 19 May 1999

Abstract IgG binding to multiple protein constituents in lysates of Jurkat cells was detected by Western blot in sera of patients with multiple sclerosis ŽMS. and systemic lupus erythematosus ŽSLE.. The distribution patterns of bands with sera tested against protein lysates from normal Jurkat cells or from Jurkat cells exposed to apoptosis or oxidative stress inducing conditions were similar in most patients, but with inter-individual differences. The number of bands with sera of both patient populations far exceeded those Ž0 or 2 bands. detected with sera of healthy controls. Proteinase K, RNase and DNase pre-treatment of cell lysates suggested a protein nature for all of the antigens and a ribonucleoprotein ŽRNP. nature for some of the antigens recognized by serum IgG of MS and SLE patients. Only two MS patients had positive anti-nuclear antibody ŽANA. titers, while all of them had positive Western blots. In addition to similarities, dissimilarities were also recognized between the humoral immune responses in MS and SLE. No IgG molecules were detected against phosphorylated proteins in the sera of MS patients, while multiple phosphoproteins were recognized by IgG molecules of SLE patients in immunoprecipitation experiments. These data suggest that in addition to ANA, the sera of MS patients contain autoantibodies directed against multiple intracellular proteins. The protein recognition patterns of immunoglobulins in MS share similarities, but also have distinct features when compared to those in SLE. The biological significance of these autoantibodies in MS remains to be understood. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Intracellular autoantigens; Autoantibodies; Multiple sclerosis

1. Introduction The etiology of multiple sclerosis ŽMS. is unknown, but an autoimmune process is postulated to be the underlying mechanism of inflammation and demyelination in the central nervous system ŽCNS.. An array of self-proteins has been investigated as the primary activation signals for T or B cells. The most intensively studied putative self-antigens with pathogenic significance are components of normal CNS myelin Žmyelin basic protein wMBPx, proteolipid lipoprotein, myelin-oligodendrocyte glycoprotein, etc.. or modified forms of these myelin proteins Žcitrullinated MBP, exon II transcript of MBP. ŽJohnson et al., 1986; Richert et al., 1989; Sun et al., 1991; Chou et al., 1992; Voskuhl et al., 1993; Martin et al., 1994; Wood et al., 1996; Genain et al., 1999.. The immune recognition of intracellular en-

) Corresponding author. Tel.: q1-215-7621898; fax: q1-215-7628404; E-mail: [email protected]

zymes Že.g., CNPase, transaldolase. and proteins with distinct sequences Žtranslated Alu repeats. in MS oligodendrocytes has also been implicated in the loss of myelin and oligodendrocytes ŽColombo et al., 1997; Archelos et al., 1998; Walsh and Murray, 1998.. A differential expression of numerous intracellularrintranuclear autoantigens Žincluding DNArRNA binding proteins. was demonstrated recently in the mRNA repertoire of MS lesions ŽBecker et al., 1997., offering at least some explanation for the increased occurrence of ANA in MS sera ŽDore-Duffy et al., 1982; Collard et al., 1997.. Whether or not there is a primary abnormality in the expression or the immune recognition of self proteins in MS remains to be investigated further ŽArchelos et al., 1998.. Many of the observed molecular alterations are secondary to the pathological process including inflammation Žoxidative damage to macromolecules. ŽVladimirova et al., 1998., apoptosis Žcleavage or phosphorylation of cellular proteins., stress Žexpression of heat shock proteins. ŽRaine, 1994. or repair Žexpression of exon II MBP.

0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 1 0 4 - 6

F. Lu, B. Kalmanr Journal of Neuroimmunology 99 (1999) 72–81

ŽCapello et al., 1997.. Oxidative damage to DNA has been demonstrated in chronic active MS lesions ŽVladimirova et al., 1998., and is thought to be related to the presence of macrophages and microglial cells which produce reactive oxygen species ŽROS. and nitric oxide ŽNO. ŽPowell et al., 1992; Bo et al., 1994; Bagasra et al., 1995; Dandona et al., 1996; Bruck et al., 1997; Vladimirova et al., 1999.. Apoptosis is believed to be involved in the elimination of infiltrating cells in plaques but probably plays little or no role in oligodendrocyte depletion ŽBonetti and Raine, 1997.. Utz et al. Ž1997. demonstrated recently that proteins selectively phosphorylated during apoptosis are recognized by ANA positive sera of patients with SLE or lupus overlap syndromes. Substrates of enzymes activated during apoptosis may be presented as modified self-proteins on the surface of tissue-specific antigen presenting cells ŽUtz et al., 1997.. In this study, we addressed the question of whether immunoglobulins in MS sera and CSF bind to intracellular proteins or modified intracellular proteins Ž‘‘neoantigens’’. related to apoptosis or oxidative stress. We generated protein enriched lysates from normal Jurkat cells and from Jurkat cells exposed to conditions which induce apoptosis Žanti-Fas, UV. or oxidative stress ŽH 2 O 2 .. Sera and CSF specimens from MS patients, healthy controls and SLE patients were tested against these protein preparations by Western blot and by immunoprecipitation in order to further characterize intracellular autoantigens in MS.

73

2. Materials and methods 2.1. Sera and cerebrospinal fluid (CSF) from patients and controls Ten CSF and serum pairs from six patients with clinically definite, laboratory supported MS ŽPoser et al., 1983., and three CSF and serum pairs from three normal controls were received from the National Neurological Research Specimen Bank in Los Angeles ŽNNRSB-LA.. From one of the six patients four CSF-serum pairs were obtained within a period of two months Ž21–4–23 days apart, respectively., and from another patient two CSF–serum pairs were obtained within 35 days. Three patients had relapsing-remitting, two secondary and one patient primary progressive course of MS. All were in a ‘‘remission’’ or ‘‘stationary’’ phase of the disease at the time of sampling Žwhich was before interferons became available. ŽNNRSBLA.. All the MS CSF specimens had an increased IgG, IgGrAlb index and oligoclonal bands ŽNNRSB-LA.. Sera of an additional four negative controls were collected from employees of the authors’ department Žgiving a total of seven normal controls.. Sera from four SLE patients, all with increased ANA, anti-dsDNA and anti-ssDNA titers ŽTable 1., were provided by the Laboratory of Microbiology and Virology, MCP-HU. All specimens were aliquoted and frozen at y708C until used.

Table 1 ANA, anti-dsDNA and anti-ssDNA titer in sera of patients and controls ANA Ž N - 1:40.

Anti-dsDNA Ž N - 40 IU.

Anti-ssDNA Ž N - 99 Urml.

SLE 29930 30041 30275 30431

1:640 Žhomogeneous. ) 1:640 Žhomogeneous. ) 1:640 Žspeckled. ) 1:640 Žspeckled.

173 751 80 832

836 1278 1666 1974

MS 6567L 6567P 6567Q 6567T 6655B 6939 6893E 6398G 6398R

Negative Negative Negative Negative Negative 1:80 Žhomogeneous and speckled. Negative Negative 1:40 Žspeckled.

Trace nuclear staining, speckled. Prominent cytoplasmic staining noted Prominent cytoplasmic staining noted, linear fibrous pattern

Controls C235 C236 C251 C254 11842-0 11898-0 11907-0

Negative 1:80 Žspeckled. Negative Negative Negative Negative Negative

74

F. Lu, B. Kalmanr Journal of Neuroimmunology 99 (1999) 72–81

2.2. Preparation of protein antigens 2.2.1. Cell culture A Jurkat cell line ŽATCC TIB 152, Clone E6-1. was grown in RPMI-1640 with glutamine ŽSigma., supplemented with 10% HI-FCS ŽSigma. and Antibiotic–Antimycotic solution ŽMediatech, Herndon, VA.. A human astrocyte cell line was kindly provided by Dr. Shoreh Amini ŽMCP-HU., and was used for protein preparation after five in vitro passages. For immunoprecipitation studies Jurkat cells were incubated at a 2 = 10 6rml concentration in a labeling medium containing 27% RPMI 1640 with phosphate, 63% RPMI 1640 lacking phosphate ŽGibco BRL, Gaithersburg, MD., 10% HI-FCS and 0.1 mCirml 32 P-labeled orthophosphate ŽDupont-NEN, Boston, MA.. 2.2.2. Generation of modified cellular proteins Labeled or unlabeled Jurkat cells were transferred to a Petri dish at 2 = 10 6rml concentration, and each culture was treated in one of the following ways: Ža. anti-Fas monoclonal antibody ŽPharmingen, San Diego, CA. at 2 mgrml concentration and protein G ŽSigma. at 1 mgrml concentration were added to the culture medium; Žb. 400 mJ UV irradiation was delivered to the culture at a distance of 9 cm for 12 s ŽStratalinker 2400; Stratagene, La Jolla, CA.; Žc. H 2 O 2 ŽSigma. was added into the culture medium at a final concentration of 10y5 M; and Žd. control Jurkat cells were incubated without any treatment. The four cultures were simultaneously processed and incubated in a CO 2 incubator at 378C for 0, 1, 2.5, 4.5, 6 and 9 h in the kinetic studies, and for 4.5 h to prepare protein lysates in the Western blot or immunoprecipitation studies. At the end of incubation, cells were collected by centrifugation and lysed. 2.2.3. Agarose gel electrophoresis of DNA to monitor apoptosis Unlabeled Jurkat cells were treated as indicated above Ža–d., and cells were collected by centrifugation at 1000 rpm for 5 min. The cell pellet was lysed Ž20 mM Tris, pH 7.4, 5 mM EDTA and 0.4% triton X-100. and incubated on ice for 15 min. DNA was extracted with phenol–chloroform–isoamyl alcohol from the supernatants after centrifugation of lysates at 14,000 rpm for 5 min at 48C ŽUtz et al., 1997.. After precipitation, DNA pellets were washed with 70% ethanol and resuspended in 10 mM Tris–HCl. Agarose gel analysis of DNA from a normal Jurkat cell culture and three differentially treated Jurkat cell cultures demonstrated increasing DNA fragmentation in the UV and anti-Fas treated samples from 2.5 to 9 h post treatment. Fig. 1a demonstrates apoptosis in the UV and antiFas treated cells and lack of apoptosis in the H 2 O 2 treated and control Jurkat cells at 4.5 h post treatment. 2.2.4. Assessment of apoptosis by flow cytometry Apoptosis of Jurkat cells treated as described above Ža–d. was determined by using an In Situ Cell Death

Detection Kit ŽBoehringer Mannheim.. Flow cytometric analysis of five thousand cells at 4.5 h post treatment revealed 69% apoptosis in the anti-Fas and 70% apoptosis in the UV treated cultures, while 3.7% and 2.8% apoptosis was detected in the H 2 O 2 treated and normal Jurkat cultures, respectively ŽCoulter, Hialeah, FL. ŽFig. 1b.. H 2 O 2 in the indicated Ž10y5 M. concentration induced no apoptosis at any time points studied. 2.2.5. Southern blot detection of oxidatiÕe damage to DNA Oxidative damage to mtDNA was quantified to estimate the overall oxidative stress to H 2 O 2 exposed cells ŽPfeifer et al., 1991, 1993; Driggers et al., 1997.. Briefly, DNA from normal, Alloxan treated Ž10 mM, 4.5 h. or H 2 O 2 exposed Ž10y5 M, 4.5 h. Jurkat cells was extracted with a QiaAmp Tissue Kit ŽQiagen, Valencia, CA.. Alloxan is known to induce oxidative damage to mtDNA, and was used to generate a positive control. After BamHI digestion, purified DNA samples were quantitated and divided into two equal aliquots. One of each pair was incubated with formamidopyrimidine DNA glycosylase ŽFPG. ŽTrevigen, Gaithersburg, MD. which introduces single strand breaks at oxidized purines. To produce single strand breaks at all abasic or sugar-modified sites in DNA, both parts of each sample pair Žnon-treated and FPG treated. were incubated with sodium-hydroxide. DNA samples were then separated by alkaline gel electrophoresis, transferred to a nylon membrane and hybridized with a biotinylated mtDNA probe wnts 14,559–15,112x ŽGibco BRL, Grand Island, NY.. Membranes were washed under high stringency conditions and developed using the PhotoGene Nucleic Acid Detection System ŽGibco BRL.. The linearized 16.5 kb mtDNA bands were measured by densitometry and the break frequency was determined by using Poisson expression Ž s s yln P0 , where s is the number of breaks per fragment; P0 is the fraction of fragments free of breaks calculated as densitometric volume of FPG q alkaliralkali treated DNA. ŽDriggers et al., 1997.. In the non-treated Jurkat cells, the proportion of mtDNA fragments free of breaks Ž P0 . was 0.96 when the FPG q alkali treated sample was compared to the alkali-treated counterpart, giving a break frequency of s s 0.038 per 16.5 fragments of mtDNA ŽFPG detected oxidative damage at purine nucleotides with a frequency of 0.038 per mtDNA fragments in normal Jurkat cells.. Both Alloxan and H 2 O 2 increased oxidative damage Žbreak frequency. in mtDNA. In the Alloxan-treated Jurkat cells, the fraction of mtDNA fragments free of breaks was P0 s 0.75 giving a break frequency of s s 0.286. In the H 2 O 2-treated Jurkat cells, the fraction of fragments free of breaks was P0 s 0.77 giving a break frequency of s s 0.258 per 16.5 kb fragments of mtDNA. 2.2.6. Polyacrylamide gel electrophoresis (PAGE) of modified proteins Control and differentially treated Jurkat cells Ža–d. were lysed by using a 2 = cell lysis buffer Ž25 mM

F. Lu, B. Kalmanr Journal of Neuroimmunology 99 (1999) 72–81

75

Fig. 1. Apoptosis of the UV and anti-Fas treated Jurkat cells. Ža. DNA fragmentation. DNA was extracted from the supernatant after centrifugation of cell lysates. In this way, the fragmented Žbut not full size genomic. DNA was preferentially collected. DNA fragmentation is demonstrated in the UV and anti-Fas treated Jurkat cells Žlanes 2 and 3, respectively., and lack of fragmentation in the H 2 O 2 treated and control Jurkat cells at 4.5 h post treatment Žlanes 4 and 5, respectively.. Lane 1 is a HindIII digested l DNA marker. Žb. Flow cytometry. Apoptosis of Jurkat cells was determined by an In Situ Cell Death Detection kit. Diagram 1: Apoptosis in 69% of anti-Fas treated Jurkat cells; 2: Apoptosis in 70% of UV treated Jurkat cells; 3: Apoptosis in 3.7% of H 2 O 2 treated Jurkat cells; 4: Apoptosis in 2.8% of non-treated Jurkat cells.

Tris–HCl, pH 8.0, 300 mM NaCl, 2% Triton X-100, 2% BSA. supplemented with Mini, EDTA-free, Complete protease inhibitor cocktail tablets ŽBoehringer-Mannheim, In-

dianapolis, IN.. One milliliter of lysis buffer was added to 5 = 10 7 cells for 60 min on ice, followed by centrifugation of lysates at 14,000 rpm. Supernatants were used for

76

F. Lu, B. Kalmanr Journal of Neuroimmunology 99 (1999) 72–81

immunoprecipitation or Western blot experiments. Proteins–lysates were separated by a 12% polyacrylamide gel ŽCrambach and Rodbard, 1971.. Unlabeled proteins were visualized in the gel by Coomassie-blue staining or transferred to a nitrocellulose membrane for immunostaining. Protein profiles of Jurkat cells treated with anti-Fas, UV and H 2 O 2 differed from those of non-treated Jurkat cells at 4.5 h post treatment ŽFig. 2.. 32 P-labeled proteins from similarly treated Jurkat cells were visualized by autoradiography, but did not show noticeable differences ŽAutoradiogram not shown.. 2.3. Western blot Following PAGE separation, electrophoretic transfer of modified proteins derived from normal, anti-Fas, UV or H 2 O 2 treated Jurkat cells was carried out as described ŽTobin et al., 1979.. For the detection of antibodies produced against modified self-proteins, nitrocellulose membrane strips were blocked with 5% BSA, washed and incubated with sera or CSF specimens of MS patients and controls. Sera and CSF specimens were diluted 1000 = and 10 = respectively, to yield a similar IgG concentration of 10–40 ugrml in all the specimens. After incubation with the primary antibody Žsera or CSF., membrane strips were incubated with an alkaline–phosphatase conjugated

anti-human IgG secondary antibody, and signals were developed by using the BM Chromogenic Western Blotting Kit ŽBoehringer-Mannheim.. 2.4. Immunoprecipitation To preclear antigens, 10 ul of Protein A Sepharose 4 Fast Flow 50% slurry ŽPharmacia Biotech, Piscataway, NJ. was added to a 200 ul volume of each cell lysate of 32 P-orthophosphate labeled Jurkat cells Ža–d., and gently mixed at room temperature for 2 h. Supernatants were collected by centrifugation at 14,000 = g for 15 s. Immunoprecipitation was carried out by adding 3.5 ul of sera or 40 ul of CSF specimens to 40 ul of precleared supernatant Ž32 P-labeled antigen.. After coupling antigens with antibodies for 2 h, 40 ul of Protein A Sepharose slurry was added to immune complexes and gently mixed at 48C for 12 h. The pellet was collected by centrifugation at 14,000 = g for 15 s, washed with the dilution buffer and with Wash buffer A Ž10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.025% Na–azide. and Wash buffer B Ž50 mM Tris–HCl, pH 6.8.. After the addition of SDS loading buffer, immunoprecipitates were boiled and loaded on a 12% SDS-polyacrylamide gel. Gels were dried and exposed for autoradiography. 2.5. ANA test Using an indirect immunofluorescent technique Žthe secondary antibody is a goat anti-human IgG and IgM fluorescein conjugate. on the HEp-2 Žhuman epithelial. cell line, determination of ANA titers was performed in the Clinical Chemistry Laboratory, Thomas Jefferson University, Philadelphia. Serum titers were determined with a screening dilution of 1:40. All samples showing a positive result at this titer were serially diluted up to 1:640 and retested to obtain a titer. Appropriate technical controls were included to avoid false positive reading of the results.

3. Results

Fig. 2. PAGE of proteins from UV, anti-Fas and H 2 O 2 treated and control Jurkat cells. Differential profiles of proteins from Jurkat cells treated with anti-Fas, UV and H 2 O 2 in comparison to that of non-treated Jurkat cells at 4.5 h post treatment Žlanes 2–5, respectively.. Note the lack of bands indicated in the normal Jurkat cell lysate Žlane 5. in the H 2 O 2 treated Jurkat cell lysate Žlane 4., and the appearance of a new band in the anti-Fas and UV treated Jurkat cell lysates Žlanes 2 and 3.. Lane 1 is a broad range SDS PAGE protein standard ŽBioRad, Pontiac, IL.. Monitoring apoptosis by agarose gel electrophoresis and by flow cytometry, and detecting oxidative damage to mtDNA by FPG treatment and Southern blot analysis Žsee Section 2. suggests that the differential patterns of proteins on PAGE are related to apoptosis in the anti-Fas and UV-treated Žlanes 2 and 3., and to oxidative stress in the H 2 O 2 -treated Žlanes 4. cell cultures.

3.1. Western blot analysis shows IgG to cellular proteins in MS sera ANA data Žcomprising antinuclear IgG and IgM. are summarized in Table 1. In addition to the high ANA titers Ž1:640 or higher. in all four SLE patients, 2 MS patients had low positive titers Ž1:40 and 1:80.. One of the 7 controls also demonstrated a low positive Ž1:80. ANA titer. Sera G and R from MS patient a 6389 showed prominent cytoplasmic staining with a negative and a positive anti-nuclear staining on HEp-2 cells, respectively. Western blots with sera of 4 SLE patients displayed individually different IgG binding patterns. Exposing blots

F. Lu, B. Kalmanr Journal of Neuroimmunology 99 (1999) 72–81

to sera from 7 negative controls Žincluding the one with positive ANA. resulted in either no band or two bands always in the same position Žan approximate MW of 45 kDa and 35 kDa.. All of the MS sera contained IgG to intracellular proteins in a poly- or oligoclonal distribution ŽFig. 3.. When sera were tested against the four lysates Žfrom anti-Fas, UV, H 2 O 2 or non-treated, control Jurkat cells. similar patterns of antibody bindings were observed in most of the quadruplets. Exception was seen with 3 sera Ž2 of which were obtained from one MS patient a6398. which showed a distinct band with a highly increased intensity and with an approximate MW of 28 kDa in the UV treated cell lysate ŽFig. 3.. Western blots with two sera ŽG and R. of MS patient a 6398 obtained one month apart seemed to be similar including the 28-kDa extra band in the UV treated protein preparation. However, serum G was ANA negative and serum R was ANA positive, both with prominent cytoplasmic staining ŽTable 1.. In contrast, Western blots with four ANA negative sera ŽL, P, Q and T, obtained within three months. from MS patient a6567 displayed high intensity staining with similar patterns when the first ŽL. and last ŽT. were tested, and low intensity staining when the second ŽP. and third ŽQ. specimens were tested ŽTable 1.. Generally, IgG detected on Western blot did not correlate with positive ANA titers in MS patients and negative controls. The ANA positive healthy control ŽC236. had negative Western blot, while all the MS patients who had negative ANA showed positive staining Žmultiple bands. on Western blots. Interestingly, the serum of MS patient a6893 was negative for both nuclear and cytoplasmic

77

staining on the HEp2 cell, but presented with the most intense staining in polyclonal distribution against Jurkat cell lysates on Western blot. Proteinase K, RNase and DNase treatment of proteins obtained from a normal Jurkat cell lysate was performed to further characterize the nature of antigens. Proteinase K pre-treatment of lysates eliminated all the bands on Western blot when tested with sera of one SLE patient, two MS patients and one negative control. RNase treatment of cell lysates eliminated some of the bands on Western blots exposed to sera of SLE and MS patients but did not change the background pattern detected with the serum of the negative control. DNase treatment did not noticeably modify the IgG binding pattern of samples on Western blots. None of the MS CSF samples Ždiluted to 10-40 ugrml IgG concentration similar to that in the adjusted serum samples. contained detectable IgG against cellular proteins from the four Jurkat cell preparations in the Western blot analysis. One of the control CSF samples showed two reproducible bands in positions also seen with some of the control sera on Western blots. The distribution pattern of IgG from some MS, SLE and normal sera was also determined against proteins prepared from non-treated and UV-treated human astrocytes. Sera containing IgG to proteins from Jurkat cells and from UV-treated Jurkat cells, were also positive with proteins from astrocytes and UV treated astrocytes. However, the IgG binding patterns against the two cell lysates revealed variances in both the high and low molecular weight regions on Western blots ŽFig. 4..

Fig. 3. Western blot analysis of serum IgG. Blots of proteins from anti-Fas, UV and H 2 O 2 treated and normal Jurkat cells Žlanes 1–4, respectively. were exposed to sera obtained from SLE patients, healthy controls and MS patients. Representative Western blots with sera of an SLE patient ŽA.; a healthy control ŽB.; and an MS patient ŽC. are shown. While healthy controls had no or two bands on Western blots ŽB., SLE and MS patients had multiple bands ŽA and C.. The four protein preparations seemed to have identical IgG binding patterns with the sera of most patients. An exception was seen with three sera from two MS patients. A band with dramatically increased intensity Ždemonstrated in lane 2, Section C. occurred in the UV-treated protein lane with an approximate molecular weight of 28 kD Žarrowhead..

78

F. Lu, B. Kalmanr Journal of Neuroimmunology 99 (1999) 72–81

Fig. 4. Comparison of serum IgG binding to proteins derived from Jurkat cells and astrocytes. Autoreactive IgG in the serum of patient a30275 recognizes both ubiquitously expressed and cell specific proteins in lymphoid and glial cells. A similar observation was made with MS sera, although with less intensive bands. Lane 1: protein lysate from Žnontreated. Jurkat cells. Lane 2: protein lysate from Žnon-treated. human astrocytes. Arrowheads indicate different antigenic targets in the Jurkat cell and astrocyte derived protein lysates.

3.2. Lack of autoantibodies against phosphorylated proteins in MS sera and CSF Immunoprecipitation was performed by using four differentially modified 32 P-orthophosphate labeled protein extracts from Žnormal, anti-Fas, UV or H 2 O 2 treated. Jurkat cells with sera of MS patients and of negative or positive controls ŽUtz et al., 1997.. IgG molecules in sera of SLE patients ŽANA G 1:640, positive anti-ds- and anti-ss-DNA. coupled with multiple phosphorylated proteins derived from the four cell lysates. Sera from neither normal controls nor from MS patients Žeither positive or negative for ANA. contained detectable levels of IgG specific for these phosphorylated proteins. Autoradiograms of immunoprecipitations were negative using CSF of both MS patients and normal controls Ždata not shown..

4. Discussion This study reveals that autoreactive IgG molecules directed against multiple intracellular epitopes of nuclear and cytoplasmic origin can be detected in the sera of MS patients. Control sera tested under similar conditions showed either no or two background bands on Western blots. In accordance with previous observations, several intracellular proteins were recognized by the sera of SLE

patients. Some of the cellular antigenic determinants recognized by IgG in MS and SLE sera are probably distributed ubiquitously, however, cell-specific differences were also noted when IgG binding to proteins from lymphocytes ŽJurkat. and astrocytes were compared. Detection of an increased level of autoreactive IgG to intracellular proteins only in sera, but not in CSF, is at variance with most of the previously described humoral immune abnormalities in MS. Antibodies against myelin and other CNS restricted antigens have been found in both sera and CSF of MS patients ŽSellebjerg et al., 1994; Warren and Catz, 1997; Colombo et al., 1997; Walsh and Murray, 1998.. A humoral abnormality predominantly detected in MS sera and very seldom in CSF includes ANA ŽCollard et al., 1997.. ANA is a diagnostic marker for SLE and overlapping autoimmune diseases. Positive ANA with low titers is found in 10-81% of MS sera depending on experimental protocols and sensitivities ŽCollard et al., 1997.. Our studies suggest that in addition to nuclear antigens, cytoplasmic antigens are also recognized by immunoglobulins in MS Žsimilar to that described in SLE.. Why these autoreactive IgG molecules present only in the sera of MS patients, when increased expression of mRNA for several nuclear and cytoplasmic proteins was detected in plaques ŽBecker et al., 1997., remains to be understood. The occurrence of these IgG molecules in MS sera indicates an immune abnormality not restricted to the CNS, although they recognize intracellular proteins in CNS cells as well. The recognition of intracellular autoantigens by helper T cells in the peripheral circulation is likely to be responsible for the polyclonal B cell activation Žas also suggested by our preliminary T cell proliferation studies.. Quantitative production of these IgG molecules seems to fluctuate over time Žas seen at least in the four sera of one MS patient a 6567. with a relatively preserved intra-individual distribution pattern on Western blot, but with interindividual differences. A longitudinal study of clinical course, gadolinium enhanced MRI and self-reactive immunoglobulins will be necessary to answer whether there is a correlation between immunoglobulin production and activity of MS. With the exception of three sera which displayed a highly intensified band in the UV treated lysate, the IgG binding patterns were generally similar when tested against the panel of four lysates Žof normal, anti-Fas, UV or H 2 O 2 treated Jurkat cells.. This observation suggests that if any modification of the normal intracellular proteins occurs secondary to apoptosis or oxidative stress in vivo in MS, the presentation of these modified proteins may be too low or too brief to mount any specific autoantibody response detectable by Western blot. Alternatively, autoantibodies may recognize both the native and modified proteins as has been described in SLE Žvon Muhlen and Tan, 1995; Utz et al., 1997.. Autoantigenic determinants seemed to include RNP in both SLE and MS. However, IgG in neither sera nor CSF

F. Lu, B. Kalmanr Journal of Neuroimmunology 99 (1999) 72–81

of MS patients bound with any of the 32 P-labeled intracellular proteins of normal, anti-Fas, UV or H 2 O 2 treated Jurkat cells in the immunoprecipitation studies. This finding indicates that phosphorylated proteins and apoptosis related phosphoproteins are not major antigenic targets in MS, in contrast to the intensive anti-phospho-protein Žbelieved to be pathogenic. response observed in SLE. Thus, despite similarities in humoral immune alterations, the underlying mechanisms of generating polyclonal or oligoclonal autoreactive immunoglobulins are likely different in SLE and MS ŽTable 2.. This observation is compatible with the partial genetic overlap between the two diseases ŽMcCombe et al., 1990; Becker et al., 1998.. The question can be raised as to the biological significance of antibodies binding to intracellular proteins. Distinct profiles of autoantibodies directed at intracellular antigens can be detected and used to facilitate differential diagnosis in various Žoften overlapping. collagen vascular disorders. They are used as prognostic markers and guide clinical or treatment monitoring. These antibodies are important tools to gain insight into cell biology, and to explore further the pathogenesis of SLE and related disorders Že.g., antibodies to apoptosis-related phosphoproteins. Žvon Muhlen and Tan, 1995, Utz et al., 1997.. However, the mechanism of production, the relatively selective target profile Že.g., nuclear antigens including ribonuclear proteins, histones and DNA, and endoplasmic reticulum associated antigens in SLE. and the exact biological role of these antibodies is not completely understood even in the best characterized systemic autoimmune diseases. ANA production in MS is usually referred to as ‘‘nonsense’’, ‘‘false-positive’’, or considered as mere evidence of dysregulation of the immune response ŽMattson et al., 1980; Aisen and Aisen, 1995; Barned et al., 1995; Mattson, 1995; Collard et al., 1997.. Although in a retrospective study a correlation was seen between ANA and age or duration of MS and a trend for correlation with disability, none of these correlations could be confirmed in a prospective study ŽMattson, 1995; Collard et al., 1997.. Only exacerbation seemed to correlate with positive ANA, supporting the previous conclusion that ANA is produced as a ‘‘by-product of systemic immune dysregulation’’ ŽCollard et al., 1997.. Autoreactive IgG molecules to multiple intracellular antigens Žas demonstrated in this study. are likely pro-

79

duced by a mechanism similar to that of ANA production in MS. We suggest that even if these antibodies are ‘‘by-products’’ of systemic immune activation, further characterization of target antigens Že.g., subgroups of RNPs. may provide useful information to better understand the pathogenesis of MS. In addition, functional Žboth protective and pathogenic. significance of these autoantibodies may be considered and tested. The autoreactive IgG repertoire in healthy individuals Žwhich may be upregulated in MS. comprises natural antibodies directed against a great variety of self and intracellular antigens ŽGuilbert et al., 1982.. Natural antibodies have been implicated in self-protective mechanisms such as blocking epitopes in damaged tissues or facilitating the clearance of self-proteins. These antibodies are part of the anti-idiotypic network with immune regulatory functions. They also have been implicated in remyelination in the Theiler’s virus induced demyelination model of MS ŽMiller et al., 1996.. At the opposite end of a functional spectrum, potentially pathogenic autoreactive IgG clones directed against intracellular proteins have been identified in MS sera and CSF. Some of these antibodies recognize autoantigens located exclusively or predominantly in oligodendrocytes Že.g., CNP, protein sequences with translated Alu repeats., suggesting their involvement in demyelination or loss of oligodendrocytes ŽColombo et al., 1997; Archelos et al., 1998; Walsh and Murray, 1998.. These observations suggest that autoantibodies to intracellular proteins may be involved in both immunological self-protection and selfdestruction. The present study adds to previous observations the finding that polyclonal IgG clones directed against intracellular proteins are present in MS sera, and are characterized by some distinct features when compared to those in SLE sera ŽTable 2.. This observation suggests that the generation of these antibodies may be related to some disease-specific mechanisms. Available data are not sufficient to determine if the increased levels of autoreactive immunoglobulins reflect an abnormal immune response, or an abnormal antigen presentation in MS. Nevertheless, the autoreactive antibody repertoire may be used to identify epitopes which are unique to or uniquely targeted in MS. Determination of the subcellular antigenic profile and clarification of the functional significance Žprotective or pathogenic features. of autoreactive IgG molecules may

Table 2 Similarities and differences in the humoral immune response of MS and SLE patients

ANA ŽIgGrIgM. ŽHep2 cells. IgG to intracellular proteins ŽJurkat cells. on Western blot Anti-RNP IgG Anti-phospho-protein IgG a

Utz et al. Ž1997..

MS

SLE a

Normal control

qry qqqrqqrq

qqq qqqrqq

yrq y

q y

qq qqqrqq

y y

80

F. Lu, B. Kalmanr Journal of Neuroimmunology 99 (1999) 72–81

contribute to a better understanding of an abnormal cell biology associated with MS.

Acknowledgements The authors are very grateful to the National Neurological Research Specimen Bank in Los Angeles for providing serum and CSF samples, and to Dr. Shoreh Amini for providing human astrocytes. The authors also thank Dr. F.D. Lublin for a critical reading of the manuscript. This study was supported by a NMSS grant RG2770A2r2.

References Aisen, M.L., Aisen, P.S., 1995. Antinuclear antibodies in multiple sclerosis. Neurology 45, 2299. Archelos, J.J., Trotter, J., Previtali, S., Weissbrich, B., Toyka, K.V., Hartung, H.P., 1998. Isolation and characterization of an oligodendrocyte precursor-derived B-cell epitope in multiple sclerosis. Ann. Neurol. 43, 14–24. Bagasra, O., Michaels, F.H., Zheng, M.Y., Bobroski, L.E., Spitsin, S.V., Fu, Z.F., Tawadros, R., Koprowski, H., 1995. Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis. Proc. Natl. Acad. Sci. USA 92, 12041–12045. Barned, S., Goodman, A.D., Mattson, D.H., 1995. Frequency of antinuclear antibodies in multiple sclerosis. Neurology 45, 384–385. Becker, K.G., Mattson, D.H., Powers, J.M., Gado, A.M., Biddison, W.E., 1997. Analysis of a sequenced cDNA library from multiple sclerosis lesions. J. Neuroimmunol. 77, 27–38. Becker, K.G., Simon, R.M., Bailey-Wilson, J.E., Freidlin, B., Biddison, W.E., McFarland, H.F., Trent, J.M., 1998. Clustering of non-major histocompatibility complex susceptibility loci in human autoimmune diseases. Proc. Natl. Acad. Sci. USA 95, 9979–9984. Bo, L., Dawson, T.M., Wesselingh, S., Mork, S., Choi, S., Kong, P.A., Hanley, D., Trapp, B.D., 1994. Induction of nitric oxide synthase in demyelinating regions of multiple sclerosis brains. Ann. Neurol. 36, 778–786. Bonetti, B., Raine, C.S., 1997. Multiple sclerosis: oligodendrocytes display cell death-related molecules in situ but do not undergo apoptosis. Ann. Neurol. 42, 74–84. Bruck, W., Bitsch, A., Kolenda, H., Bruck, Y., Stiefel, M., Lassmann, H., 1997. Inflammatory central nervous system demyelination: correlation of magnetic resonance imaging findings with lesion pathology. Ann. Neurol. 42, 783–793. Capello, E., Voskuhl, R., McFarland, H.F., Raine, C.S., 1997. Multiple sclerosis: re-expression of a developmental gene in chronic lesions correlates with remyelination. Ann. Neurol. 41, 797–805. Chou, Y.K., Bourdette, D.N., Offner, H., Whitham, R., Wang, R.Y., Hashim, G.A., Vandenbark, A.A., 1992. Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis. J. Neuroimmunol. 38, 105– 113. Collard, R.C., Koehler, R.P.M., Mattson, D.H., 1997. Frequency and significance of antinuclear antibodies in multiple sclerosis. Neurology 49, 857–861. Colombo, E., Banki, K., Tatum, A.H., Daucher, J., Ferrante, P., Murray, R.S., Phillips, P.E., Perl, A., 1997. Comparative analysis of antibody and cell-mediated autoimmunity to transaldolase and myelin basic protein in patients with multiple sclerosis. J. Clin. Invest. 99, 1238– 1250. Crambach, A., Rodbard, D., 1971. Polyacrylamide gel electrophoresis. Science 172, 440–450.

Dandona, P., Thusu, K., Cook, S., Snyder, B., Makowski, J., Amstrong, D., Nicotera, S., 1996. Oxidative damage to DNA in diabetes mellitus. Lancet 347, 444–445. Dore-Duffy, P., Donaldson, J.O., Rothman, B.L., Zurier, R.B., 1982. Antinuclear antibodies in multiple sclerosis. Arch. Neurol. 39, 504– 506. Driggers, W.J., Holmquist, G.P., LeDoux, S.P., Wilson, G.L., 1997. Mapping frequencies of endogenous oxidative damage and the kinetic response to oxidative stress in a region of rat mtDNA. Nucleic Acids Res. 25, 4362–4369. Genain, C.P., Canella, B., Hauser, S.L., Raine, C.S., 1999. Identification of autoantibodies associated with myelin damage in multiple sclerosis. Nat. Med. 5, 170–175. Guilbert, B., Dighiero, G., Avrameas, S., 1982. Naturally occurring antibodies against nine common antigens in human sera: I. Detection. Isolation and characterization. J. Immunol. 128, 2779–2787. Johnson, D., Hafler, D.A., Fallis, R.J. et al., 1986. Cell-mediated immunity to myelin-associated glycoprotein, proteolipid protein, and myelin basic protein in multiple sclerosis. J. Neuroimmunol. 13, 99–108. Martin, R., Whiteker, J.N., Rhame, L., Goodin, R.R., McFarland, H.F., 1994. Citrulline-containing myelin basic protein is recognized by T-cell lines derived from multiple sclerosis patients and healthy individuals. Neurology 44, 123–129. Mattson, D.H., Ross, R.P., Arnason, B.G.W., 1980. Isoelectric focusing of IgG eluted from multiple sclerosis and subacute sclerosing panencephalitis brains. Nature 287, 335–337. Mattson, D., 1995. Antinuclear antibodies in multiple sclerosis. Neurology 45, 2300–2301. McCombe, P.A., Chalk, J.B., Pender, M.P., 1990. Familial occurrence of multiple sclerosis with thyroid disease and systemic lupus erythematosus. J. Neurol. Sci. 97, 163–171. Miller, D.J., Njenga, M.K., Murray, P.D., Leibowitz, J., Rodriguez, M., 1996. A monoclonal natural antibody that promotes remyelination suppresses central nervous system inflammation and increases virus expression after Theiler’s virus-induced demyelination. Int. Immunol. 8, 131–141. Pfeifer, G.P., Drouin, R., Riggs, A.D., Holmquist, G.P., 1991. In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine Ž6-4. pyridone photoproducts by ligation-mediated polymerase chain reaction. Proc. Natl. Acad. Sci. U.S.A. 88, 1374–1378. Pfeifer, G.P., Drouin, R., Holmquist, G.P., 1993. Detection of DNA adducts at the DNA sequence level by ligation-mediated PCR. Mutation Res, 288, 39–46. Poser, C.M., Paty, D.W., Scheinberg, L. et al., 1983. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann. Neurol. 13, 227–231. Powell, T., Sussman, J.G., Davies-Jones, G.A.B., 1992. MR Imaging in acute multiple sclerosis: ringlike appearance in plaques suggesting the presence of paramagnetic free radicals. AJNR 13, 1544–1546. Raine, C.S., 1994. The Dale E. McFarlin memorial lecture: the immunology of the multiple sclerosis lesion. Ann. Neurol. 36, S61–S72. Richert, J.R., Robinson, E.D., Deibler, G.E., Martenson, R.E., Dragovic, L.J., Kies, M.W., 1989. Evidence for multiple human T cell recognition sites on myelin basic protein. J. Neuroimmunol. 23, 55–66. Sellebjerg, F.T., Frederiksen, J.L., Olsson, T., 1994. Anti-myelin basic protein and anti-proteolipid protein antibody-secreting cells in the cerebrospinal fluid of patients with acute optic neuritis. Arch. Neurol. 51, 1032–1036. Sun, J., Link, H., Olsson, T., Xiao, B.G., Anderson, G., Ekre, H.P., Linington, C., Diener, P., 1991. T and B cell responses to myelinoligodendrocyte glycoprotein in multiple sclerosis. J. Immunol. 146, 1490–1495. Tobin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354. Utz, P.J., Hottelet, M., Schur, P.H., Anderson, P., 1997. Proteins phosphorylated during stress induced apoptosis are common targets for

F. Lu, B. Kalmanr Journal of Neuroimmunology 99 (1999) 72–81 autoantibody production in patients with systemic lupus erythematosus. J. Exp. Med. 185, 843–854. Vladimirova, O., O’Connor, J., Cahill, A., Alder, H., Kalman, B., 1998. Oxidative damage to DNA in plaques of MS brains. Mult. Scler. 4, 413–418. Vladimirova, O., Lu, F., Shawver, L., Kalman, B., 1999. The activation of protein kinase C induces higher production of reactive oxygen species by mononuclear cells in patients with multiple sclerosis than in controls. Inflammation Res. Žsubmitted.. von Muhlen, C.A., Tan, E.M., 1995. Autoantibodies in the diagnosis of systemic rheumatic diseases. Semin. Arthritis Rheum. 24, 323–358. Voskuhl, R.R., McFarlin, D.E., Tranquill, L.R., Deibler, G., Stone, R., Maloni, H., McFarland, H.F., 1993. A novel candidate autoantigen in a multiplex family with multiple sclerosis: prevalence of T-lympho-

81

cytes for an MBP epitope unique to myelination. J. Neuroimmunol. 46, 137–144. X X Walsh, M.J., Murray, J.M., 1998. Dual implication of 2 ,3 -cyclic nuX cleotide 3 phosphodiesterase as major antigen and C3 complementbinding protein in the pathogenesis of multiple sclerosis. J. Clin. Invest. 101, 1923–1931. Warren, K.G., Catz, I., 1997. The effect of intrathecal MBP synthetic peptides containing epitope P85 VVHFFKNIVTP98 on free anti-MBP levels in acute relapsing multiple sclerosis. J. Neurol. Sci. 148, 67–68. Wood, D.D., Bilbao, J.M., O’Connors, P., Moscarello, M.A., 1996. Acute multiple sclerosis ŽMarburg type. is associated with developmentally immature myelin basic protein. Ann. Neurol. 40, 18–24.