The intrathecal synthesis of virus-specific oligoclonal IgG in multiple sclerosis

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J o u r n a l of Neuroimmunology

ELSEVIER

Journal of Neuroimmunology 54 (1994) 75-80

The intrathecal synthesis of virus-specific oligoclonal IgG in multiple sclerosis C.J.M. Sindic *, Ph. Monteyne, E.C. Laterre Laboratory of Neurochemistry and Department of Neurology, Catholic University of Louvain, Cliniques Universitaires Saint-Luc, 53-59 Avenue Mounier, 1200Brussels, Belgium Received 1 March 1994; revision received and accepted 17 June 1994

Abstract

A highly sensitive antigen-mediated capillary blot technique was developed for the detection of virus-specific oligoclonal IgG in paired CSF and serum samples from patients with various neurological diseases. In multiple sclerosis, intrathecal synthesis of oligoclonal antibodies was present against measles (70%), rubella (60%), varicella zoster (40%) and mumps (30%); in most cases (75%), such synthesis involved two or more viruses. In contrast, antibodies against a non-neurotropic virus (cytomegalovirus) were rarely produced in CSF from MS patients (5%). However, this 'polyspecific' reaction was not restricted to MS samples but was also observed in neurolupus and in the late phase of infectious diseases of the central nervous system. These anti-viral antibodies could be produced without de novo replication of the corresponding viral genome and are likely mere bystanders of an ongoing immune response.

Keywords: Intrathecal immunity; Intrathecal IgG synthesis rate; Oligoclonal bands; Multiple sclerosis; Infections of the central nervous system; Neurotropic virus

I. Introduction

The presence of oligoclonal immunoglobulin G (IgG) bands restricted to the cerebrospinal fluid (CSF) is the hallmark of the intrathecal humoral immune response observed in a very high proportion (approx. 95%) of patients with multiple sclerosis (MS) (Laterre et al., 1970; Thompson et al., 1979; Laurenzi et al., 1980; Keir et al., 1990). However, all attempts at defining MSspecific antigens responsible for this humoral immune response have been unsuccessful. It is still not clear whether these oligoclonal I g G are linked to a viral infection, an autoimmune reaction or some other manifestation of hypersensitivity unrelated to these putative MS-specific antigens. Thus, the relevance of the CSF-restricted oligoclonal I g G to the pathogenesis of the MS process remains obscure. The long-term persistence within the CNS of m e m ory B cells after a viral brain infection has been reported in a murine model (Gerhard and Koprowski,

* Corresponding author. Fax: ( + 32-2) 764 3679 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 5 7 2 8 ( 9 4 ) 0 0 0 9 2 - 3

1977). Measles, rubella, varicella zoster (VZ) and m u m p s infections are very common during childhood. These neurotropic viruses probably affect the brain more frequently than clinically observed (Gibbs et al., 1959) and could induce the recruitment of specific B cells within the CNS and their persistence as memory ceils. The influx of activated T-helper cells across the b l o o d - b r a i n and the b l o o d - C S F barriers seems to play a key role in initiating the MS process and coud lead to the re-activation of memory B cells present in the CNS. Such a re-activation could result in the production of antibodies against various neurotropic infectious agents and this production has been previously called 'polyspecific' (Felgenhauer and Reiber, 1992). The early report by A d a m s and Imagawa (1961) that serum antibody to measles is raised in MS patients has been confirmed by numerous studies. The mean serum titres in MS are more than twice those in controls, but in individual cases this increase has no diagnostic value. Similarly, high anti-measles activity was also observed in the serum of patients with chronic hepatitis, infectious mononucleosis and systemic lupus erythematosus (Laitinen and Vaheri, 1974; Triger et al., 1976). The

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C.J.M. Sindic et al. /Journal of Neuroimmunology 54 (1994) 75-80

finding of a lowered ratio of serum to CSF anti-measles antibodies in 57% of MS patients suggested to Norrby and colleagues (1974a) a local synthesis. The same authors also found intrathecally produced antibodies against rubella, mumps and herpes simplex in 19%, 15% and 11% of the patients, respectively (Norrby et al., 1974b). Local production to at least one of these viruses was found in 71% of patients, to two viruses in 48% and to three in 16%. A higher frequency of local synthesis of such antibodies was observed more recently by the use of sensitive enzyme immunoassays giving continuous concentrations (in arbitrary units instead of titer steps) in both the CSF and the serum simultaneously tested at the same IgG concentration (Reiber and Lange, 1991; Felgenhauer and Reiber, 1992). These locally produced antibodies migrated in electrophoresis as oligoclonal bands, as shown by Nordal et al. (1978) and Vartdal et al. (1980). These bands were detected by an imprint immunofixation technique requiring the concentration of CSF samples which may lead to loss of proteins a n d / o r aggregation of immunoglobulins. In addition, the successive imprints from a single gel could reduce the specificity, sensitivity and resolution of this technique. In this work, we avoid these potential difficulties by means of a highly sensitive antigen-mediated capillary blot technique after isoelectric focusing of the unconcentrated CSF and the paired serum samples (Bukasa et al., 1988; Depr6 et al., 1988; Boucquey et al., 1990; Sindic et al., 1990). Using this technique, we analyse the polyspecific production of CSF antibodies against measles, VZ, rubella and mumps antigens in patients with MS and other various neurological disorders. In addition, we look for the occurrence of CSF antibodies against a non-neurotropic virus, the cytomegalovirus (CMV), in MS cases.

1980). These two patients had no systemic inflammatory disease and in our hands, acute disseminated encephalomyelitis is not associated with CSF-restricted oligoclonal IgG (Sindic, 1994). At the moment of CSF sampling, eight patients were relapsing but recovered during the follow-up, three displayed a relapsing progressive form of the disease, four had a primary progressive course and five were stable. None received immunosuppressive treatment. Group III ( n - - 3 ) consisted of two patients with neurolupus and one with the Guillain-Barr6 syndrome (GBS). In the first case of neurolupus, the presenting symptoms were characterized by relapsing transverse myelitis and an attack of optic neuritis associated with anti-DNA and anti-SSA serum antibodies. The second case suffered from systemic lupus for 4 years and developed a radiculomyelitis at the level Th4-Thl0 in January 1992. A few months later, she presented a left optic neuritis. Her antinuclear factor was homogeneously positive at 1/160. The GBS case developed his disease even though being under intense immunosuppression after a renal graft, and presented a systemic CMV infection with positive cultures from urine and saliva. Group IV (n--14) consisted of patients with various infectious diseases of the central nervous system (CNS): four with VZ meningitis (isolated meningitis in one case, two cases associated with zoster ophthalmieus and one with zoster spinalis), three with herpetic encephalitis, one with rabies encephalitis, one with progressive multifocal leukoencephalopathy (this patient displayed CSFrestricted oligoclonal IgG bands late in the course of his disease), two with neuro-AIDS (a patient with CNS toxoplasmosis and a patient with AIDS-related dementia), one each with neuroborreliosis, neurosyphilis and tuberculous meningitis.

3. Materials and methods 2. Patients

We studied four groups of patients (n = 49). No data related to their immunization status were available. The first group (n = 12) was used as a control and consisted of 11 non-neurological patients suffering from minor neurosis or tension headache and one patient with cervicarthrosis myelopathy. Group II (n = 20) consisted of 20 patients with MS. All displayed oligoclonal IgG bands restricted to the CSF. Diagnosis was clinically definite in 15, and laboratory-supported definite in three patients (Poser et al., 1983). The remaining two patients displayed unilateral monosymptomatic retrobulbar neuritis and were considered as possible MS because of the simultaneous presence of periventricular hypersignals by magnetic resonance imaging (MRI) and of CSF-restricted oligoclonal IgG bands, but dissemination in time was not present (Bauer,

3.1. Samples All paired samples of CSF and serum were collected for diagnostic purposes and aliquots were stored at -20°C. IgG in CSF and serum were assayed by immunoturbidimetry (Turbitimer, Behring, Marburg, Germany). Detection of CSF-restricted oligoclonal IgG bands was performed by an immunoaffinity-capillary mediated blot technique (Sindic and Laterre, 1991).

3.2. Antigens Measles, VZ, CMV, herpes virus hominis and mumps virus antigens for complement fixation tests, rubella antigens for hemagglutination inhibition tests, Borrelia burgdorferi antigens as well as the correspond-

C.J.M. Sindic et al. /Journal of Neuroimmunology 54 (1994) 75-80

ing control antigens were from Whittaker Bioproducts (WalkersviUe, MD).

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4. Results

4.1. Control antigens 3.3. Antisera Rabbit anti-human IgG and alkaline phosphataseconjugated rabbit anti-human IgG specific for gammachain were obtained from Dako (Copenhagen, Denmark) (code A 090 and D 336, respectively). 3.4. Immunoaffinity-mediated capillary blotting Agarose gel plates (1 x 110 × 240 mm) were prepared in 30 ml distilled water with 0.36 g agarose IEF (Pharmacia) and 4.3 g sorbitol (Merck) containing 2 ml ampholine pH 3.5-9.5 (Pharmalytes, Pharmacia). For the study of the total IgG pattern, CSF and sera were diluted with distilled water to obtain a uniform IgG concentration of 5/.~g/ml; 10 p~l of paired samples, i.e. 50 ng IgG, were then applied side by side on the anodic part of the gel and isoelectrically focused for 70 min at 10°C in a LKB Multiphor Unit. The gel was then blotted onto a polyvinylidene difluoride (PVDF) sheet (Immobilon, 0.45 /~m, Millipore, Bedford, MA) previously coated overnight by the rabbit anti-IgG (gamma-chains) antiserum at a concentration of 10 /zg/ml (1 ml/10 cm 2 of PVDF area). The immunoaffinity-mediated capillary blotting was performed under a uniform weight of 1 kg for 40 min at 10°C. It was assumed that agarose gels were more suitable than polyacrylamide gels for rapid and efficient capillary diffusion of proteins onto the PVDF sheet (polyacrylamide gels should require electroblotting). The immunoblot was then washed in Tris buffer saline (TBS, Tris 20 raM, NaCl 500 mM, pH 7.5) containing 0.1% Tween 20 (Technicon, Tarrytown, NY) and afterwards dipped into a solution of 0.25% glutaraldehyde (Merck) in PBS for 20 min at 4°C. The immunoblot was further washed in TBS-Tween before incubation for 60 min at 20°C with alkaline phosphatase-conjugated rabbit anti-human IgG antiserum diluted 1000 times in TBS containing 0.3% defatted milk powder. After three 15-min washings with TBS, the immunoblots were stained using an alkaline phosphatase conjugate substrate kit (Bio-Rad, Richmond, CA). Within the same isoelectrofocusing run for detecting the total IgG pattern, 10/zl of both undiluted C S F and the corresponding serum diluted to the same IgG concentration were applied to the gel. However, these were transferred onto a P V D F sheet previously coated overnight by the viral antigens under study, diluted 10 times in TBS. The immunoaffinity-mediated blotting and the staining were performed as described above.

Immunoblots coated with control antigens prepared from non-infected cells remained uniformly negative for all samples under study (results not shown). 4.2. Patients without inflammatory disorders of the CNS (control group; n = 12) Our technique failed to detect immunostaining of the blots in native CSF (and in the corresponding serum samples diluted to the same IgG concentration as the CSF) in 5, 7, 3 and 7 of the 12 control cases against measles, rubella, VZ and mumps antigens, respectively (Table 1). In all but one of the remaining cases, a mirror pattern of oligoclonal IgG antibodies was present in both CSF and serum samples at the same pH and with the same staining intensity. In the one exception (a patient suffering from tension headache), CSF displayed faint oligoclonal anti-VZ antibodies not detectable in the corresponding serum (Fig. 1). An intrathecal synthesis of anti-VZ antibodies was thought to occur in this case. 4.3. Patients with MS (n = 20) Only two patients out of 20 (10%) displayed either no immunostaining or a mirror pattern for the antigens from the four neurotropic viruses (measles, VZ, rubella and mumps) under study. Interestingly, these two patients were considered as possible MS because they presented with only an attack of optic neuritis associated with CSF-restricted oligoclonal IgG and periventricular hypersignals on MRI scans. CSF-restricted oligoclonal antibodies were detected against four anti-

Table 1 Virus-specific oligoclonal IgG in the CSF from controls and MS patients Not detectable

Mirror pattern (stronger serum reactivity)

CSF-restricted oligoclonal antibodies (%)

Controls (n = 12)

Measles Rubella Varicella zoster Mumps

5 7 3 7

7 (0) 5 (0) 8 (0) 5 (0)

0 0 1 (8%) 0

1 5 3 4

5 (1) 3 (3) 9 (5) 10 (3)

14 (70%) 12 (60%) 8 (40%) 6 (30%)

Multiple sclerosis (n = 20)

Measles Rubella VariceUa zoster Mumps

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CO(-) CSF

pH 8,7

S

PML CSF

GBS S

CSF

COl*) S

CSF

NEUROLUPUS

S pH 87

CSF

S

CSF

S

CSF

S

CSF

S

8.3 813

8.0

810

7.L

7L 69

VZ-coated PVDF

Fig. 1. Immunoaffinity-mediatedcapillary blotting of anti-VZ antibodies in CSF and serum (S) tested at the same IgG concentration. Under normal conditions, the immunoblots either were unstained (no detectable antibodies: CO(-) in the first two lanes) or revealed the presence of oligoclonal anti-VZ IgG antibodies in both CSF and serum (mirror pattern). Here, this mirror pattern is exemplified by a case of GBS and excluded any intrathecal antibody synthesis. The serum sample of a case of PML contained stronger anti-VZ oligoclonal IgG bands than the CSF as a result of an intrathecal synthesis of IgGs with other specificities. Only one control patient displayed surprisingly faint but undoubtful anti-VZ oligoclonal bands specific to the CSF (CO(+), lanes at the left). gens in three patients, against three antigens in one (measles, zoster and rubella) (Fig. 2), against two antigens in 11 (measles and rubella in six, measles and zoster in two, measles and mumps in one, rubella and zoster in one, zoster and mumps in one) and against one antigen in three (measles, rubella and mumps in one case each). The local production of anti-measles IgG antibodies was thus the most frequent, occurring

MULTIPLE

CSF

pH

S

CSF

S

SCLEROSIS

CSF

S

CSF

6.0 IgG

MEASLES

VZV

RUBELLA

Fig. 3. Representative capillary blotting of IgG and anti-measles, VZ

and rubella antibodies in CSF and serum (S) from a case of neurolupus. CSF-restricted oligoclonal IgG and antibodies against the three antigens studied were clearly detectable, indicating an intrathecal polyspecific immune reaction. in 14 of the 20 cases (70%) (Table 1). Intrathecal production of anti-rubella, anti-VZ and anti-mumps antibodies was present in 12 (60%), eight (40%) and six (30%) cases, respectively. In contrast, only one MS CSF displayed a slight intrathecal production of antiCMV antibodies. By comparison with the CSF, some serum samples contained a greater number of, or more strongly immunostained, oligoclonal IgG antibodies against measles (one case), rubella (three cases), VZ (five cases) and mumps (three cases) antigens (Table 1). This indirectly indicated the presence in the CSF of IgGs with other specificities, resulting in a lower proportion of antibodies against the antigen under study. A similar observation was made in patients with infectious diseases of the CNS (see Fig. 1, anti-VZ antibodies in a case of PML).

S

4.4. Patients with other inflammatory diseases of the nervous system (n = 3)

8.7

8.3

In addition to the presence of CSF-restricted oligoclonal IgG bands, the two patients with neurolupus also displayed a polyspecific intrathecal immune response against the measles, VZ and rubella antigens under study in one case (Fig. 3), against V Z antigens in the other. In contrast, the GBS patients had numerous oligoclonal IgG bands and antibodies in both CSF and serum ('mirror pattern') without detectable intrathecal production of antibodies against measles, rubella, VZ, mumps and CMV antigens (Fig. 1).

8.0 7.4 6.9 6.0 IMMUNOBLOTS

IgG

MeasLes

VZV

RubeLLa

Fig. 2. Representative capillary blotting of IgG and anti-measles, VZ and rubella antibodies in CSF and serum (S) from a MS patient. The presence of CSF-restricted oligoclonal IgG and antibodies against the three antigens under study demonstrated intrathecal synthesis.

4.5. Patients with infectious diseases of the CNS (n = 14) Three out of four patients with VZ meningitis displayed an intrathecal production of specific antibodies;

C.J.M. Sindic et aL /Journal of Neuroimmunology 54 (1994) 75-80

VZ MENINGITIS

CSF

pH

CSF

S

CSF

S

i

8.7 8.3 8.0 7.L 6.9 6.0 MEASLES

VZV

RUBELLA

Fig. 4. Representative capillary blotting of anti-measles, VZ and rubella antibodies in CSF and serum (S) from a case of VZ meninitis associated to zoster ophthalmicus. Note the strong intrathecal synthesis of anti-VZ oligoclonal IgG antibodies, the presence of four anti-measles oligoclonal IgG bands and the negative reaction against rubella antigens.

the negative results for the fourth case could be due to an early sampling in the course of the disease. The three patients with herpetic encephalitis were characterized by the intrathecal production of anti-Herpes simplex antibodies as well as of anti-VZ antibodies, as the consequence of the well-known serological crossreactions between both viruses. Antibody production unrelated to the causal infectious agent was observed in four cases: against measles antigens in a case of V Z meningitis (Fig. 4), against V Z antigens in a case each of neuroborreliosis and of neuro-AIDS complicated by brain toxoplasmosis, and against mumps antigens in a case of herpetic encephalitis. It should be noted that in the first two cases the samples under study were collected 3 months after onset of the disease and specific treatment, and that in the last case the CSF sample was collected 10 years after the acute disease at the occasion of an epileptic fit (an intrathecal synthesis of oligoclonal anti-herpes simplex virus antibodies was still present in this sample).

5. Discussion

The main result of this investigation is to demonstrate that most MS patients (18 out of 20, 90%) displayed an intrathecal production of antibodies against one or more of the four neurotropic viruses under study. In fact, these antibodies were present in all cases with clinically definite or laboratory-supported clinically definite MS. Only the two 'possible' MS

79

patients, with dissemination in space but not in time, were negative. These locally produced antibodies were directed at least against two antigens in 15 cases (75%) and occurred most frequently against measles (14 out of 20, 70%). It should be noted that our prevalence rates of intrathecal synthesis were very close to the ones reported by Felgenhauer and Reiber (1992) in a study of the antibody index calculated from enzyme immunoassays. They found an intrathecal synthesis in 79% of cases for measles, in 70% for rubella and in 62% for VZ, whereas our figures are 70, 60 and 40%, respectively. These anti-viral antibodies displayed an oligoclonal pattern which did not correspond to the major oligoclonal IgG bands detected in the same CSF sample after the same run of isoelectric focusing. Such a polyspecific reaction was not restricted to CSF samples from MS patients but was also present in two cases of neurolupus and, at a lesser degree, in one case of neuro-AIDS and in three cases of infectious diseases of the CNS after clinical recovery. In the latter cases, however, the bulk of the CSF oligoclonal I g G bands was directed against the causal infectious agent (VZ, herpes simplex and Borrelia burgdorferi, respectively). Local synthesis of anti-measles, anti-herpes and antirubella antibodies has also been detected in mumps meningitis, even if most I g G bands were directed against mumps antigens (Vandvik et al., 1982). Only one patient from the control group (n = 12) displayed a faint intrathecal synthesis of anti-VZ antibodies which was however clearly detectable on the immunoblots. This female patient suffered from headache and asthenia and the extensive neurological examination remained negative. No cutaneous zoster lesions had been reported or were present at the time of the lumbar puncture and routine analysis of the CSF was normal. The presence of CSF B cells secreting specific anti-VZ antibodies in the absence of pleocytosis has been reported during cutaneous zoster (Forsberg et al., 1986) as well as the occurrence of V Z meningitis without cutaneous lesions (Mayo and Booss, 1989). In absence of direct proof of a viral infection in this case (for example, by P C R detection of the viral genome within the CSF), no definite conclusion may be drawn on this unexpected occurrence of intrathecal production of anti-VZ antibodies. In conclusion, this polyspecific intrathecal immune response has to be considered as a side-product of a chronic immune stimulation within the CNS. Its relationship to the etiopathogenesis of MS seems to be weak. These anti-viral antibodies are likely mere 'bystanders' of the ongoing immune response and could be produced without some de novo replication of the corresponding viral genome (Godec et al., 1992; Nicoll et al., 1992). Our negative results in the two cases with isolated retrobulbar neuritis suggest that dissemination

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C.J.M. Sindic et al. /Journal of Neuroimmunology 54 (1994) 75-80

in time of the demyelinating lesions is required for the production of such antibodies. The use of this polyspecific reaction for diagnostic purposes must take into account its occurrence not only in MS but also in chronic CNS infections and in other inflammatory CNS disorders. However, the continuous synthesis of these antibodies in absence of their antigenic target may be linked to the same mechanisms which are responsible for the very chronic immune activation observed in the MS process (Tourtellotte et al., 1980).

Acknowledgements The authors are thankful to Prof. C.J. Pfau for critical reading of the manuscript and to Mrs. M.P. Van Antwerpen for skillful technical assistance. This work was supported by grants from the "Groupe beige d' Etude de la Scl6rose en plaques", "Fondation B6n6" and "Kiwanis-Club Centre-Ardennes".

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