evidence for bound antineuronal antibodies in brains of nzb/w mice

Journal of Neuroimmunology, 38 (1992) 1472154 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

JNI 02182

147

Evidence for bound antineuronal antibodies in brains of N Z B / W mice

Patricia M. Moore

Department of Neurology, Wayne State University School of Medicine, Detroit, MI, USA

(Received 15 May 1991) (Revised, received 8 October 1991 and 9 January 1992)

(Accepted 10 January 1992)

Key words: Neuronal systemic lupus erythematosus; Antibody; Brain elution

Summary

Antibodies reactive with neuronal tissue are present in the sera of the murine models of systemic lupus erythematosus (SLE). Access of these antibodies to the central nervous system is an important prerequisite to the hypothesis that these antibodies affect neuronal function. In this study, we isolated antibodies from neutral and acid washes of brain parenchyma of NZB/W F 1 mice. Antibody could be eluted from the brains of NZB/W F 1 but not control mice. The immunoglobulin was predominantly IgGt; the binding characteristics of the brain eluted antibody were narrower than those of antibody from sera and eluted from visceral organ.

Introduction

Murine and human systemic lupus erythematosus (SLE) exemplify autoantibody and immune complex mediated tissue dysfunction. Antineu- ronal antibodies and immune complex deposition within the CNS are well described in human SLE but the murine systems are less well defined (Wilson et al., 1979; Bluestein et al., 1981; Schwartz and Roberts, 1983; Toh and Mackay, 1981; How et al., 1985; Bonfa et al., 1987; Hanly et al., 1988). Early observations included immune complex deposition in the choroid plexus of

Correspondence to: P.M. Moore, Wayne State University, School of Medicine, Department of Neurology, 421 E. Can- field Ave., 3124 Elliman Building, Detroit, MI 48201, USA.

NZB/W F 1 mice (Lampert and Oldstone, 1973). Recently, murine antineuronal antibodies in the serum have been demonstrated by cytotoxicity to cultured cerebellar cells (Harbeck et al., 1978) and Western blots of brain plasma membranes (Narendran and Hoffman, 1989). Work in my laboratory shows that immunoglobulins isolated from MRL/lpr and NZB/W but not Balb/c mice specifically bind to neuroblastoma and brain cortex plasma membranes (Moore, 1990).

Although immunoglobulins reactive with neuronal tissue occur in the NZB/W F 1 mouse sera they are unlikely to directly affect neuronal function unless they are present in the central nervous system. In some human neuroimmunological diseases such as subacute sclerosing panencephalitis (SSPE) and multiple sclerosis, antibodies bound to brain can be eluted from thoroughly washed brain homogenate by acid elution (Matt-

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son et al., 1982; Bernheimer et al., 1983; Weil et al., 1979). Acid elution yields antibodies bound to kidneys in murine SLE but has not been studied in the brains of these animals. This study investi- gates the recovery of acid elutable antibody from the brains of N Z B / W F l mice.

Materials and methods

Animals Female N Z B / W F 1 hybrid and Balb/c J mice

were purchased from Jackson Laboratories and cared for in an approved animal care facility. In two separate experiments, groups of mice were ether anesthetized and terminally bled. In the first experiment brains and livers were removed from 18 10-month-old Balb/c and N Z B / W mice. In the second experiment, 20 brains, kidneys and livers frozen and stored at -70°C for less than 2 months were used. The ages of mice in this group ranged from 8 to 12 months and were equally mixed in autoimmune and control mice.

Immunoglobulin elution The procedure was similar to that of Gilden

(Gilden et al., 1978). The organs were minced, homogenized, and washed extensively overnight in cold PBS (pH 7.1) with 0.1% Na azide. The mixture was centrifuged for 20 min and the pellet was resuspended in PBS and centrifuged three additional times. The supernatants were all saved. The pellets were resuspended in 10 ml 0.5 M acetic acid and 0.15 M NaC1 (pH 2.8) containing 1 p~g/ml pepstatin and 0.004 M E-amino-caproic acid and within 2 min neutralized with 6 N K2CO 3. All samples were precipitated with 40% ammonium sulfate and dialysed extensively against cold PBS for 48 h. The protein concentrations were determined by Lowry assay.

The class of immunoglobulin was determined by dot blot. Triplicate samples of undiluted, 1 : 10, and 1 : 100 eluates were absorbed on TBS wetted nitrocellulose for 6 h, controls were normal mouse IgG, IgM, and IgA (CappeD. The nitrocellulose was washed (TBS-Tween), blocked with 3% dry skimmed milk (DSM) for 1 h and again washed. Horseradish peroxidase (HRP) conjugated goat

anti-mouse IgG, IgM, or IgA (1 : 250 Capped was added. After 1 h the nitrocellulose was again washed with TBS-Tween and developed for color with BCIP/NBT substrate (Biorad).

Immunoglobulin purification The neutral washes and acid eluates were pu-

rified over an anti-mouse IgG sepharose column (Pierce). The column was washed with buffered saline and the sample was incubated with IgG- sepharose for 1 h. The column was washed with 10-fold volume of PBS and the effiux collected in 1-ml fractions. Acetic acid pH 3.2 was added to the column, the fractions were collected and im- mediately neutralized. The samples were concentrated (Amicon filter) and the IgG concentration estimated at OD280 in an LKB spectrophotome- ter. The subclasses of IgG from the tissue samples were determined as above for immunoglobulins except HRP conjugated goat anti-mouse IgG1, IgGza , lgGzb , and IgG 3 (Southern Biotechnology) were used in the ELISA.

Antibody binding activity

ELISA. Standard ELISA techniques were used. The targets were calf thymus DNA (25 /zg/well), cultured hepatoma or neuroblastoma cells and fetal neurons. Hepatoma and neuroblastoma (N2a) cells (American Tissue Culture Col- lection, Bethesda, MD; (1 × 105/well)) were grown to confluency in 96-well microtiter plates (Costar) in Dulbecco's MEM supplemented with 5% fetal calf serum and 1 mM glutamine, then air dried and stored at room temperature. Fetal neurons were obtained by dissecting fetal Balb/c brains at 15 days gestation. Using a dissecting microscope, the cortices were isolated from 8 to 10 fetuses and separated into individual cells by repeat gentle pipetting in glass Pasteur pipettes. Cells were cultured in 96-well microtiter plates in Dulbecco's high glucose MEM supplemented with 10% FCS and 5% horse serum. After 3 days in culture, the cells were washed gently and fixed with acetone for 5 min. The ELISAs were performed by washing the plates with PBS, blocking for 1/2 h in 3% BSA, incubating with 50 /xg of sample immunoglobulin for 3 h, washing in PBS- Tween three times, incubating with HRP conju-

gated goat anti-mouse IgG light chains (Cappel) for 1 h and again washing three times with PBS- Tween. The substrate O-phenylenediamine was added to each well and the plates read within 30 min at a wavelength of 450 nm in a Titertek ELISA reader. PBS background values were subtracted from sample values before analysis.

Western blots. Plasma membrane preparations of brain were prepared according to the method described previously (Poduslo et al., 1986). Briefly, cortices from 10-15 B a l b / c mice were quickly removed after the animals were killed by cervical dislocation. Single-cell suspen- sions were prepared by incubation of the cortices in dispersion medium (0.1% trypsin, 1 mM EDTA, 280 mM sucrose, 10 mM glucose, 20 mM NaPO4, 10 mM Hepes; p H 7.5) on ice for 30 min. The supernatant was decanted and centrifuged at 300 x g. for 10 min. The undissolved particles were resuspended in dispersion medium and the above steps repeated. The pellets from the collected supernatants were washed and repelleted in Dul- becco's medium. The cells were then lysed in hypotonic medium in a glass cell homogenizer and the nuclear components removed by centrifu- gation. The crude membrane preparat ion was purified on a discontinuous sucrose gradient. The band at the 0.25/0.85 M interface was collected. Plasma membrane proteins were separated by 9.6% SDS-PAGE. 50 /zg protein were placed in each lane and the gel electrophoresed at 50-60 V for 16 h. The proteins were transferred to nitrocellulose sheets at a constant power of 180 mA in transfer buffer (20 mM Tris-HC1; pH 8.2) 150 mM glycine, 20% methanol in a Biorad Transblot cell. Completeness of protein transfer was determined by staining both the gel and a strip of nitrocellulose with Coomassie blue. The nitrocellulose was cut into strips and stored until ready for use. The strips were then wetted with TBS- Tween and blocked with 3% dry skim milk for 1 h and then lightly washed. Each strip was incubated with 20 /xg of IgG isolated from the eluates and incubated for 16 h. The strips were then washed three times in TBS-Tween and then incubated with AP conjugated goat anti-mouse IgG for 2 h. After two subsequent washes with TBS-Tween, substrate B C I P / N B T was added. At optimal color

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TABLE 1

ANTINEURONAL ANTIBODIES IN NZB/W F 1 MICE.

ELISA screening of NZB/W and Balb/c mouse strains. ELISA was performed as detailed in text using 1 : 80 dilution of serum. Assays were performed in triplicate and PBS background subtracted from number before analysis. End point optical densities were read at 450. PBS background was not greater than 0.185 in these assays. In the first section, any sample with mean OD greater than 0.35 was considered to have antibodies reactive with the target; the same samples were reanalysed with only OD greater than 0.8 were considered positive.

DNA Liver Neuro- blastoma

OD > 0.35 NZB/W 16/50 40/50 34/50 (32%) (80%) (68%)

Balb/c 0/25 4/25 (16%) 2/25 (8%)

OD > 0.8 NZB/W 10/50 26/50 21/50 (20%) (52%) (42%)

Balb/c 0/25 0/25 0/25

development the reaction was stopped and the strips were dried.

Results

Presence o f neuron reactive antibodies in the sera o f N Z B / W F 1 mice

Serum samples from 50 female N Z B / W F 1 mice, aged 9-11 months, were tested for antibodies reactive with nervous system tissue by ELISA on neuroblastoma cells. The same samples were also tested by ELISA for reactivity with calf thymus D N A and liver cells. The results, shown in Table 1, indicate the presence of neuron reactive antibodies in the serum of 68% of N Z B / W F 1 mice when an optical density of 0.35 was considered positive. Using a higher OD ( > 0.8) as a cutoff point reduced the number of normal mice with autoantibody to zero, while 42% of the N Z B / W mice still had antibody reactive with neuroblastoma cells. The predominant subclass was IgG2a. Because the sera of these mice con- tain polyclonal antibodies to cytoplasmic, nuclear, and membrane structures it is not possible to further define the specificity of these samples with the immunoglobulins employed for this study.

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Isolation of immunoglobulins from brain tissue Immunoglobulins could be recovered from tis-

sue homogenates. Cold saline washes of brain and liver or liver/kidney yielded both IgG and IgM. Extensive washing of the tissue with saline brought forth minimal detectable immunoglobulin, acetic acid washes of the same tissue revealed only traces of immunoglobulin in the normal mouse tissue, both IgG and IgM in the N Z B / W liver and only IgG in the N Z B / W brain. The IgG was purified by affinity column and the quantity of IgG recovered from each of the samples is listed in Table 2. Equivalent amounts of IgG per gram of tissue were recovered from neutral washes of brain and visceral organs. A smaller amount of immune bound IgG was obtained from brain than from visceral organs. Multiple isotypes of IgG were present in neutral washes of all tissue and acid elution of visceral organs. How-

TABLE 2

RECOVERY OF IgG FROM ORGANS

Overnight washes and acid eluates of tissues were passed over an anti-mouse IgG sepharose column as described in text. IgG was eluted from the column and concentrated by centrifuga- tion in an Amicontube.

Organs IgG recovery total/xg (/xg/g tissue)

Expt. 1 Expt. 2

NZB/W Brain neutral wash 900 (120) 720 (90)

NZB/W Brain Acid eluate 80 (10.6) 91 (11.4)

NZB/W Liver (and kidney) neutral wash 810 (121) 780 (99)

NZB/W Liver (and kidney) Acid eluate 130 (19.4) 112 (14.2)

Balb/c Brain neutral wash 90 (11.2) 84 (12.5)

Balb/c Brain acid wash N.D. a N.D.

Balb/c Liver (and kidney) neutral wash 120 (13.3) 98 (11.8)

Balb/c Liver (and kidney) acid wash 7 N.D.

a N.D., not detectable; limit of detection 0.5 p.g.

TABLE 3

SUBCLASS OF IGG IN IMMUNOGLOBULIN ISOLATES.

NZB/W Brain neutral wash IgG 1 IgG2a IgG2b NZB/W Brain acid eluate IgG I (2nd exp. IgG 1 and IgG2a) NZB/W Organ neutral wash IgG D IgGza, IgGzb, IgG 3 NZB/W Organ acid eluate IgGza, IgG2b

ever, IgG l was the major subclass in acid elutions from brain in both experiments (Table 3).

Binding characteristics of recovered immunoglobulins

The immunoglobulins isolated from each tissue by neutral and acid elutions were evaluated for antibody activity by ELISA using several different targets. (Table 4). Neutral washes from all organs were comparable to serum in binding to DNA and liver cells in the ELISA. However, in N Z B / W mice, acid eluates of visceral organs also showed reactivity with DNA and hepatoma targets, but acid eluates of brain showed greater binding to neuroblastoma and fetal brain cells than to DNA or liver cells. Comparisons of binding to neuroblastoma and fetal brain cells are more difficult because the background of the fetal brain cells was always higher.

Immunoglobulins from the neutral and acid eluates from brains of' N Z B / W and Balb mice

TABLE 4

ELISA OF RECOVERED IMMUNOGLOBULINS ON DNA, LIVER CELLS, NEUROBLASTOMA CELLS, AND FETAL BRAIN CELLS.

These were averages of replicate samples because there was insufficient material to perform the assay in triplicate.

Immunoglobulin Target from DNA Liver Neuro- Fetal

cells blastoma brain

NZB/W Brain neutral wash 0.25 0.38 0.41 0.65

NZB/W Brain acid elution 0.09 0.19 0.34 0.57

NZB/W Liver neutral wash 0.31 0.27 0.22 0.53

NZB/W Liver acid elution 0.18 0.21 0.26 0.48

Balb/c Brain neutral wash 0.0 0.07 0.05 0.14

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were also tested on Western blots of Ba lb /c brain plasma membranes as shown in Fig. 1. 20 /~g/ml of immunoglobulin from the neutral wash of tissues from Ba lb / c mice did not bind to plasma m e m b r a n e proteins, al though im-

1 2 3 4 5 6

53

39

29 23

Fig. 1. Western blot of recovered immunoglobulins on brain cortex plasma membranes. For preparation of plasma membranes and details of procedure see text. Primary antibody 20 /~g each lane: lane 1, Balb/c brain neutral wash; lane 2, NZB/W brain neutral wash; lane 3, NZB/W brain acid eluate; lane 4, Balb/c liver neutral wash; lane 5, NZB/W

liver neutral wash; lane 6, NZB/W liver acid eluate.

munoglobulins from both the neutral washes and acid eluates of visceral organs and brain from the N Z B / W F 1 mice did bind. Immunoglobulins from acid elutions of N Z B / W brains bound to 23, 29, 39 and 53 kDa proteins, while those from neutral washes of the same brains bound only to 23 and 29 kDa proteins. Immunoglobulins from the neutral wash of the liver bound to three proteins of slightly different size than the immunoglobulin from the neutral wash of brain. The acid eluates of liver did not bind to the brain plasma membranes. In a previous analysis, serum from N Z B / W mice bound to brain plasma membranes of apparent molecular masses 101, 68, 63, 57, 53, 43, 39 and 31 kDa (Moore, 1990).

Discussion

This study demonstrates that antibodies can be eluted from brain in N Z B / W F 1 mice. The isolation of the immunoglobulins from brain demonstrates that antibodies in these mice have access to or are produced in the central nervous system. This is an important first step in investigating whether these antibodies are pathogenic. No- tably, isolated immunoglobulins differ in predominant isotype and binding characteristics from antibodies eluted from liver and k i d n e y s of N Z B / W F 1 mice. The difference in binding between antibodies eluted from brain and from other organs supports the hypothesis that the brain bound antibodies are a distinct population with potential pathogenic effects on the nervous system.

The presence of autoreactive immunoglobulins in low titer in the sera of some Ba lb / c mice is considered normal (Steele and Cunningham, 1978; Haspel et al., 1983). although autoreactive antibodies in normal mice are seldom persistent and do not induce disease. Immunoglobulin could be acid eluted from brain only in the mice with autoimmune disease. This is not a reflection of the quantity of the immunoglobulin present but represents immunoglobulin bound to tissue (Weil et al., 1979; Mattson et al., 1982; Bernheimer et al., 1983). Immunoglobulins present in the neutral washes of all organs represent those in the intravascular space, thus the hypergammaglobu-

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linemia of the N Z B / W F~ animal is reflected in the increased quantity of immunoglobulin in the neutral washes of all organs. Previous studies of acid elution of immunoglobulins in murine models of SLE have used renal tissue. Much of this antibody eluted from the kidney has represented immune complex deposition (Slack et al., 1984). In our study less immunoglobulin could be eluted from brains than the viscera. Recovered immunoglobulins were notably IgG from the brain although both IgG and IgM could be recovered from the liver or l iver /k idney preparations.

IgG~ was present disproportionately in the brains of the N Z B / W F1 animals compared with their sera or eluates from visceral organs. Al- though the IgG~ is the predominant subclass in normal animals, IgGza is quantitatively greater in the N Z B / W F1 sera and is the major subclass of autoreactive antibodies in both sera and tissue (Slack et al., 1984; Fisher et al., 1988). IgG~ it was present as a smaller percentage of the total immunoglobulin from eluates than from the serum. Thus, the presence of IgG 1 in the brain does not represent a general tissue-binding property of the isotype. Nor is there any evidence that IgG~ crosses the b lood-bra in barrier more easily than other antibody subclasses. Supporting the possible significance of our observation is the finding that IgG~ is the most prominent isotype in experi- mentally induced (typically to a viral infection) IgG production in the murine central nervous system (Griffin, 1981). Identification of IgG 1 as the predominant isotype in the brain may provide an explanation for the paucity of histological changes in neuro-SLE in mice and perhaps in humans. Variations in the biological properties of tissue bound antibodies determine their ability to fix complement and induce inflammation. Be- cause IgG~ in the mouse fixes complement poorly, its deposition is not associated with striking inflammation. Thus, the antibody may bind to the cell surface, effect changes in cellular function, and yet, induce minimal histological changes. In the first elution, IgG~ was the only Ig isotype identified; in the second elution it was present along with IgG2a. It is unlikely that freezing and storing was responsible for the increased recovery of one isotype. The more plausible explanation is that among the pooled brains were several that

were distinctly unusual in either the presence or the absence of the IgG2a, thus distorting the results of the group. It is not currently possible to test this using individual brains because the quantity of immunoglobulin recovered from each has been too small for evaluation.

Notably, the antibody binding characteristics of the brain eluted antibody differed from that of serum or eluted from visceral organs. The widest spectrum of binding activity occurred, as ex- pected, in the serum. We have shown that serum auto-antibodies from these mice do bind to plasma membrane proteins in Western blots. Al- though the 53-kDa and 39-kDa proteins were identified by antibodies in both serum and brain elutes, the 23- and 29-kDa proteins identified by eluted antibodies were not identified by serum antibodies. Possible explanations include antibodies binding to the 23- and 25-kDa protein are not present in the serum or are present in such low concentrations they are not detectable by our current Western blot techniques (Moore, 1990). It is difficult, however, to compare the binding characteristics of immunoglobulins from neutral and acid washes because the low pH elution procedure causes partial denaturation of IgG antibody (Weil et al., 1975). Thus binding activity is most appropriately compared with acid-eluted antibody from other tissues. Antibody eluted from brain showed greater reactivity to neuronal and less binding to non-neuronal targets than antibody eluted from liver or kidneys. Three different types of nervous system tissue were used as targets for antibodies. The neuroblastoma cells have the advantage of being a single cell type but the disadvantages of being neoplastic are more closely identified with peripheral nervous tissue. Fetal cortical cells are primarily neuronal but vascular and glial elements may be present and could account for the high background activity. Brain cortex plasma brain, like the fetal brain cells, are central nervous system tissue, and in addition are primarily cell surface rather than cytoplasmic or nuclear antigens. The plasma membranes are also primarily but not purely neuronal. Use of the three targets together is complementary and comparison with the DNA and liver target supports the statement that these cells bind to neuronal tissue.

The origin of the brain-eluted antibody is not known. Immunoglobulin within the CNS may be from serum or intrathecal in origin. Because antineuronal antibodies are present in the serum, it is frequently assumed that they arise in the serum and cross the blood-brain barrier. It has been postulated that immune complex disease may al- ter the permeability of the blood-brain barrier and allow access of macromolecules (including brain reactive antibodies) to the CNS. Studies of animal models of acute immune complex disease reveal that abnormalities do occur but primarily in the blood-CSF barrier, not blood-brain barrier. Using rabbit serum albumin, investigators reported changes in CSF IgG and albumin, but not in the calculated extravascular space of rab- bits (Hoffman et al., 1983). Another group of investigators using bovine serum albumin to study chronic immune complex disease in rats did de- scribe increased radiolabel in the brain parenchyma (Peress et al., 1977). Recent studies suggest that specific antibody may cross into the central nervous system either through neurons projecting outside the blood-brain barrier or by specific transport mechanisms (Ritchie et al., 1986; Fabian and Petroff, 1987; Rodriguez and Lennon, 1990). Non-specific binding of antibody by Fc receptors in the choroid plexus also occurs and provides another potential route of access (Peress et al., 1981). Alternately, CNS immunoglobulins could arise from resident plasma cells within the CNS. Histological studies do show some, if minimal, inflammation although im- munochemical analysis of these ceils is not yet available (Rudick and Eskin, 1983). To date, both the quantities of antibody and the number of inflammatory cells within the CNS have been too small to analyse the origins of these antibodies.

Ultimately it is the identification of the epitopes for these antibodies within the CNS that will permit further studies of the antibodies within the CNS. From these experiments it is apparent that antibody binding to nervous system antigens is enriched in the antibodies recoverable from brains compared to the serum immunoglobulins. At least some of the epitopes are in plasma membranes as determined by Western blot. Al- though we cannot conclude that the epitopes are neuronal proteins, the plasma membrane prepa-

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ration is enriched for neuronal antigens compared with glial and vascular (Poduslo et al., 1986). The binding of the eluted antibody to neuroblastoma cells and fetal brain cells in ELISA also supports a predominant neuronal specificity of the antibody. The relevance of these antibodies will depend upon determining an effect of these antibodies on neuronal function. Currently, at least, we can identify neuron-reactive antibodies within the CNS of NZB/W F t mice.

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evidence for bound antineuronal antibodies in brains of nzb/w mice

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