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Multiple sclerosis immune complexes An analysis of component antigens and antibodies.

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Multiple Sclerosis Immune Complexes:
An Analysis of Component Antigens
and Antibodies
P. K. Coyle, MD, and Zora Procyk-Dougherty, MS
Serum immune complexes were isolated from 21 patients with multiple sclerosis and 23 control subjects, who were
normal or had other neurological diseases, and then probed for neurotropic viral and brain-reactive components.
Multiple sclerosis complexes contained antibody to herpes simplex (13 cases), measles (8 cases), cytomegalovirus ( 5
cases), and rubella virus ( 5 cases). In some complexes, herpes simplex or cytomegalovirus antigen was detected along
with antibody. Myelin basic protein or antibody to myelin basic protein was found in the complexes of 7 patients with
multiple sclerosis and 2 patients with other neurological diseases. Serum complexes containing antibody reactive with
galactocerebroside and ganglioside were present in 12 patients with multiple sclerosis and 3 with other neurological
diseases, Over 60% of the multiple sclerosis group had IgM and IgA serum complexes, including 5 patients with very
high IgA complex levels. This study suggests that patients with multiple sclerosis have ongoing systemic virus
production with resultant immune complex formation. In addition, they often have serum complexes that contain
brain-reactive components.
Coyle PK, Procyk-Dougherty 2: Multiple sclerosis immune complexes: an analysis of component antigens
and antibodies. Ann Neurol 16:660-667, 1984
Multiple sclerosis (MS) is a neurological disorder
whose pathogenesis and cause remain elusive despite
intensive research efforts. The two leading hypotheses
are that MS is primarily an infectious disorder, implicating one or more viral agents {l5, 291, and that it
is an immune-mediated disorder against an unknown
antigen within the central nervous system [18, 32, 331.
It is likely that both elements are critical to the development of MS.
Immune complex (IC) formation is a hallmark of
both infectious and autoimmune disorders [ 19, 301.
Previous investigators using different multiple assays
have found circulating IC to be present in up to 70%
of patients with MS {1, 8, 10, 13, 14, 22, 26-28].
Very few data are available, however, on the nature of
these complexes and their component antigen and
antibody. Based on the premise that appropriate ICs
will be formed if MS is either infectious or autoimmune in nature, we have examined the components of
serum ICs from MS and non-MS subjects.
Materials and Methods
Subjects
Forty-four subjects were studied; the only criterion for selection was a detectable level of serum IC. The subjects included 21 patients with clinically definite MS [23] and 23
From the Department of Neurology, Health Sciences Center, T-12,
State University of New York at Stony Brook, Stony Brook, NY
11794.
subjects without MS: 6 normal controls and 17 controls with
other neurological diseases (4 patients with polyradiculopathy, 3 patients with seizure disorder, and 1 patient each with
Alzheimer’s disease, congenital myotonia, essential tremor,
herpes simplex encephalitis, mental retardation, peripheral
neuropathy, polymyositis, rubella vaccine reaction, subacute
sclerosing panencephalitis [SSPE], and syphilis). None of
the patients with MS had other medical conditions that give
rise to IC formation with the possible exception of one patient with a history of frequent staphylococcal skin infections. Five patients with MS had never received any treatment, 16 had received steroids in the past, and one of these
had been treated with azathioprine and steroid agents. None
had received any treatment for at least three months prior
to IC testing. Blood was obtained by venipuncture, and the
serum aliquoted and stored at either - 20 or - 70°C. All serum samples were negative for rheumatoid factor as determined by latex fixation (RA Screening Tests; Tago, Burlingame, CA). Student’s t test for unpaired samples was used
for statistical analysis of the data.
IC Assay and Isolation
Complexes were detected using a modified Raji cell enzymelinked immunosorbent assay (ELISA). Raji cells (1 x lo6)
were fixed to flat-bottomed wells of a microtiter plate (Falcon
plates; Becton-Dickinson, Oxnard, CA) using poly-~-lysine
and glutaraldehyde [ll]. As in the standard assay 1311,
serum specimens were diluted 1:3 with phosphate-buffered
Received Nov 7, 1983, and in revised form Mar 12, 1984. Accepted
for publication Mar 28, 1984.
Address reprint requests to Dr Coyle.
saline solution (PBS), p H 7.4, and preincubated with a complement source for 30 minutes at 37°C. Samples were then
added to each of triplicate wells (50 p1 per well). Cohn fraction I1 IgG (Miles Scientific, Naperville, IL) was diluted in
PBS to a concentration of 50 mg/ml and then heat aggregated at 62.5"C for 40 minutes. Insoluble aggregates were
removed by centrifuging at 2,000 rpm for 30 minutes. The
concentration of aggregated human globulin (AHG) in the
supernatant was measured by spectrophotometry, diluted to
I mg/ml, and kept in small aliquots at - 70°C. Prior to use,
an aliquot of AHG was thawed, diluted with PBS to give a
series of concentrations ranging from 3 to 125 pg/ml, and
then run in the assay. After 1 hour at 37"C, plates were
washed three times in PBS-Tween 20 (0.1%).Then 100 p1
of horseradish peroxidase (HRP)-conjugated antihuman
IgG diluted in ELISA buffer (5% chick serum-PBS-Tween)
was added to each well. After 30 minutes' incubation at
room temperature in the dark, plates were again washed
three times in PBS-Tween. Enzyme substrate solution (2.3
mM orthophenylenediamine in 0.24 M citrate, 0.05 M phospate p H 5 with 0.03% hydrogen peroxide) was added (125
per well). The enzyme substrate reaction was allowed to
continue for 15 minutes in the dark at room temperature.
After 100 p.1 from each well was transferred to a second
plate, the reaction was stopped by the addition of 50 p.1 of
I N sulfuric acid per well, and the absorbance read at 488 nm
on a Dynatech microELISA reader (Dynatech Labs, Alexandria, VA). The mean optical density (OD) reading of the
AHG was used to generate a straight-line standard curve,
which typically spanned a range from 0.3 to 1.6 OD units.
Serum samples were read from this in micrograms per milliliter of AHG, then corrected by the dilution factor. To be
considered a positive, sample levels had to be at least 2
standard deviations above the mean for a group of 10 negative controls.
ICs were isolated by elution from Raji cells as previously
reported {>I.Serum was incubated with Raji cells (7.5 x lo7
cells per 1 ml of sera) for 1 hour at 37°C. Raji cells were then
washed at room temperature three times in PBS and centrifuged for 5 minutes at 1,400 rpm to remove the serum.
Bound complexes were eluted and dissociated by the addition of 1 ml of chilled 0.1 M sodium borate, p H 10. After 10
minutes at 37"C, the cells were quickly sedimented by centrifugation at 2,000 rpm and the eluates collected and stored
without neutralization at 4 or -20°C. Previous work had
indicated that antigens and antibodies are dissociated by this
technique (51.
lmmu noglobulin Assay
IC isolates were examined for IgG, IgM, and IgA content
using ELISAs. Affinity-purified goat antihuman IgG, IgM, or
IgA (Tago) was used to coat microtiter wells at 1 pg/ml. IC
samples were diluted and added to triplicate wells in 25 p1
amounts. Known amounts of purified human immunoglobulins (Cappel Laboratories, West Chester, PA) were run to
generate a standard curve. Samples were incubated for 2
hours at room temperature. Plates were washed three times,
and then 100 pl of the appropriate HRP-antihuman immunoglobulin antisera was added to each well. After 1 hour
at room temperature, plates were washed and enzyme substrate added. After a IS- to 30-minute incubation in the
dark, the reaction was stopped with sulfuric acid and the
plate read at 488 nm. The mean absorbance of the known
amounts of immunoglobulin was used to generate a standard
curve. The test samples were read from this in nanograms
per milliliter and then corrected by the dilution factor. Assay
sensitivity was 1 ng/ml of immunoglobulin, with interassay
and intra-assay variablility less than 10%.
Viral Antigen and Antibody Assay
Antibodies to herpes simplex virus (HSV), cytomegalovirus
(CMV), measles, and rubella were measured in ELISAs. Viral or control antigens (Flow Laboratories, McLean, VA)
were diluted 1 :200 and used to coat alternate rows of the
microtiter plate. IC isolates were added to duplicate viral and
duplicate control wells, and the ELISA was carried out as
indicated previously for immunoglobulin. A set of positive
antisera and of negative antisera were included in each assay
as positive and negative controls. The absorbance of the IC
samples was read as the mean OD of viral wells minus the
mean OD of control wells. ICs were read as positive for
specific viral antibodies if their absorbance was greater than 3
standard deviations above the mean of the negative controls.
Samples gave consistent results on multiple assays.
ELISAs were used to detect type 1 HSV (HSV1) and
CMV viral antigens. Mouse monoclonal antibodies to the
P40 to 45 nucleocapsid proteins of HSV1, along with agents
for ascites control, were purchased from Cappel Laboratories. Monoclonal antibody to the GP58 protein to CMV was
a kind gift of D r Arnold Levine (Department of Microbiology, Stony Brook). Alternate rows of a microtiter plate were
coated with viral-specific monoclonal antibody or control. IC
samples were added, and the assay was run as indicated, with
one additional step. One hundred microliters of diluted human antiserum to either HSV or CMV (Flow Laboratories)
was added to each well for 1 hour prior to the HRP conjugate. Commercial HSV or CMV antigens were run as positive controls; a series of five IC isolates without specific viral
antibodies were run as negative controls. Positive IC samples
were interpreted as in the antibody assay. The variation
among replicate samples was less than 10%; all positive samples were run several different times and gave consistent
results.
Brain Antibody and Antigen Assay
Antibody activity directed against myelin basic protein
(MBP), galactocerebroside, and ganglioside was measured by
ELISA. Human MBP (Cal-Med, South San Francisco, CA)
was used to coat microtiter wells at 1 pg/ml. Bovine brain
galactocerebroside (C-87 52; Sigma Chemical Co, St. Louis,
MO) was first dissolved in 1 N hydrochloric acid, then diluted and coated at 100 pgiml. Bovine brain ganglioside (G2250; Sigma Chemical Co) was coated at 100 pg/ml. IC
samples were applied to duplicate wells. Because there was a
degree of nonspecific IgG binding with all three antigens,
a known amount of IgG greater than that found in the
IC isolates was run along with control isolate (PBScomplement added to Raji cells). The assay was carried out
as described, except that HRP-antihuman IgG (Fab)z was
used as conjugate rather than as whole IgG. Background
binding was less than 0.1 OD units. A positive result was
determined by a mean absorbance above that of the IgG and
Coyle and Procyk-Dougherty: MS Immune Complexes 661
I5O
“
A
>
W
r
I
501
.+.
.
W
-J
H
U
.
.
.I
....
.*
8
I
.
.
.
.....
....
i%
.
.
5001
/
300
0
-
.
-
.
.
E
\
Cyl
.
200 -
C
v
Cyl
H
-
100 -
1
ON0
(N.6)
(N.17)
MS
.
..: ..! .. .
.. .
.
.i
.. .
.
0
0.
.
control isolate. Replicate samples gave a variation of less than
1593, and all positive results were confirmed in at least two
different assays.
MBP antigen was measured by ELISA. Mouse monoclonal
antibodies reictive with residues 130 to 137 of human MBP
or ascites control (Hybritech, San Diego, CA) were used to
coat alternate rows of a microtiter plate. IC isolates were
added to duplicate wells in both rows. Known amounts of
MBP were included in the assay. Following 2 hours at room
temperature, plates were washed and an optimal dilution of
rabbit anti-MBP serum (Cal-Med) was added to each well.
After a 1-hour incubation and washings, this addition was
followed by HRP-antirabbit IgG (Miles Scientific). The assay was then completed by the addition of enzyme substrate
as described. Samples were read as the mean absorbance of
MBP wells minus the mean absorbance of control wells.
Concentrations of MBP in the IC samples were read from a
standard curve generated from the known amounts of MBP
run in the assay. The assay was able to detect 1 ng/ml of
MBP. Intraassay variation was less than lo%, and all positive
samples were confirmed on at least two separate assays.
Results
Serum IC levels in AHG varied from 45 to 90 pg/ml
in the normal controls, 38 to 130 pdml in patients
with OND, and 32 to 146 pg/ml in the patients with
MS (Fig 1). There was no significant difference in the
mean level of IC among the three groups (69, 72, and
68 pg/ml, respectively).
After elution from the Raji cell surface, IC samples
were examined for IgG, IgM, and IgA content (Fig 2).
1
1
*
*
0
0.
1
.
*
0
Fig I. Serum immune complex (IC) levelsfor the positive control
subjects, patients with other neurological diseases (OND),and
patients with multiple sclerosis (MS) were measured in a
modiJZedRaji ceit! assay. Levels were expressed as micrograms per
milliliter of aggregated human globulin (AHG) equivalent.
Samples were interpreted as positive based on an A H G level
greater than 2 standard deviations above the mean of a negative
control group (ten samples without IC activity). Absolute levels of
IC were comparable in all three groups (mean levels of 69%72,
and 68 pglml, respectively).
..
.
.
8!
(N:21)
*
*
*
*
+ CONTROL’S
0.0
1
1
1
: i
’
1
*
.
1
L
@
-J
CON ON0 MS
CON OND MS
161 1f7J 1Zf)
151 1f2J ff6J
CON O N 0 MS
(f1 171 (f3J
I9G
L---T----J
I9M
I9A
’
-+
ISOLATED IC
Fig 2. Isolated immune complexes (IC)from the three groups were
examinedfor IgG, IgM, and I g A content. There was no signifcant difference in IgG recwered, with a mean value of 101 ngi’ml
from control (CON) complexes, 141 nglml from subjects with
other neurological diseases (OND), and 120 nglml from complexes of patients with multiple sclerosis (MS). IgM was detected
in complexes of 5 controls, 12 OND subjects, and 16 MS subjects. I g A was detected in complexes of I control, 7 OND, and 13
MS subjects. (Numbers in parentheses refer t o numbers of subjects .)
All three groups showed a wide variation in amount of
complexed IgG. Complexed IgM was detected in 5
control, 12 OND, and 16 MS subjects. IgA complexes
were found in only 1 control, 7 OND, and 13 MS
subjects. Five of the patients with MS showed very
high levels of complexed IgA (1300 ng/ml). Their
corresponding serum IgA levels were somewhat low,
70 to 235 mddl (normal range, 61 to 330 mg/dl).
Complexed immunoglobulin was not highly correlated
with serum levels (Fig 3). To calculate monomeric immunoglobulin binding to Raji cells, sera negative for
JC but with elevated immunoglobulin levels (I@,
2,600 mg/dl; IgM, 310 mg/dl; IgA, 435 mg/dl) were
bound and eluted from Raji cells and the eluate tested
for immunoglobulin. Only 0.25% of free IgG, 0.96%
of free IgM, and 0.33% of free IgA were bound and
eluted.
IC samples were tested for IgG antibodies to four
common viral agents (Table 1). None of 6 normal controls had serum complexes containing viral antibodies.
Among the OND controls, 1 subject showed complexes with antibodies to HSV and CMV.This was a
662 Annals of Neurology Vol 16 No 6 December 1984
Table 1. Isolated Immune Complexes: Viral-sbecificAntibodv
Group
HSV
Measles
CMV
Rubella
~~~~
Controls
(n = 6)
Patients with
non-MS
neurologic
disease
(n = 17)
Patients with
0
0
0
0
1
2"
1
lb
13
8
5
5
a!
0
a
I
0
100
200
MS
,
1
300
400
(n = 21)
"Included a patient with subacute sclerosing panencephalitis (measles
slow virus infection).
bRubella vaccinee.
300
a
a
no
HSV = herpes simplex virus; CMV = cytomegalovirus; MS =
multiple sclerosis.
0
0
200
400
/
0
a0
a n
0
!'
a.
:*
50
0
a
-
100
500
150
0
a
)a
0
a
n CONTROL
o
0
100
ON0
-
200
COMPLEXED
300
a
800
Ig (ng/rnl)
Fig 3. Levels of immunoglobulin isolated from immune complexes
(abscissa) were plotted against corresponding serum IgG, IgM,
and 1gA levels (ordinate)for the three groups. Bars refer to normal rangesfor serum. There was no direct correlation between
serum and complex levels, particularly in the cases of I g M and
IgA. (MS = patients with multiple sclerosis; OND = subjects
with other neurological diseases.)
patient with partial complex seizures and a history of
status epilepticus precipitated by episodic and unexplained fevers. The patient with SSPE had complexes
containing measles antibody, as did 1 patient with a
psychiatric seizure disorder; the rubella vaccinee also
had complexes containing rubella antibody. Among
the 2 1 patients with MS, 13 had serum complexes with
antibody directed against HSV. Measles antibody was
detected in 8 of the MS complexes, CMV antibody in
5, and rubella antibody in 5. All antibodies were in the
IgG class; no IgM or IgA antiviral activity was detected.
The IC samples were tested along with a series of
positive controls (sera with high viral-specific antibody
titers) as well as a series of negative controls. As outlined in Table 2, the absorbance reading of positive
MS complexes was consistent with the presence of
viral antibody. Complexed vird antibody, just as in the
case of complexed immunoglobulin, was not correlated with corresponding serum levels. Three patients with MS who had complexed CMV antibody
had no free serum antibody, 2 patients with complexed
measles antibody had no free antibody, and 2 patients
with complexed rubella antibody had no free antibody.
IC samples that were positive for HSV and CMV
antibodies were then examined for specific viral antigen (Table 3). Complexes from 6 patients with MS
contained HSVl antigen along with antibody; 2 patients with MS had complexes with C&iV antigen and
antibody.
In total, 16 to 21 patients with MS (76%) had serum
IC with viral antibody to at least one of the four agents
tested (Table 4). Four patients (19%) had complexes
containing antibodies to three agents, 7 (33%) had
antibodies to two agents, and 5 (24%) had antibody to
a single agent. No correlation was apparent between
viral-specific IC and level of IC, type of MS, duration
of disease, clinical disease activity, or disability status.
IC samples were also examined for brain-reactive
components (Table 5). Seven MS complexes contained
antibody to MBP; only 2 non-MS complexes (from
patients with herpes encephalitis and Guillain-Bard
syndrome) showed antibody to MBP. When these MS
complexes were examined for MBP, 5 of the 7 had
detectable antigen levels, ranging from 20 to 175 ng
per milliliter of MBP. Twelve of the MS ICs contained
Coyle and Procyk-Dougherty: MS Immune Complexes 663
Table 2. Enzyme-Linked Immunosorbent Assay for Viral Antibody"
Antibody
Positive
Control Serab
Negative
Control Serab
HSV
0.153 t 0.069
0.028
0.022
(n = 9)
0.026 5 0.023
(n = 8)
0.028 t 0.014
(n = 5 )
CMV
0.181 5 0.066
(n = 4)
Measles
Rubella
0.218 5 0.116
(n = 3)
0.143 -+ 0.059
(n = 3)
*
(n =
6)
0.030 -+ 0.024
(n =
6)
MS Immune Complexes
0.170
?
0.094'
(n = 13)
0.189
5
0.060'
(n = 5)
0.269
?
0.132'
(n = 8)
0.184 2 0.046'
(n = 5 )
"All values are expressed as mean optical density readings +- 1 standard deviation, read at 488 nm.
bControl sera positive or negative for specific viral antibody were run at 1:400 dilution.
'Significantly different from negative controls (p < 0.001).
Abbreviations as in Table 1.
Table 3. Enzyme-Linked lmmunosorhent Assay for Viral Antigena
Antigen
Positive Virus
Controlb
Negative Control
Immune Complexes
Positive MS
Immune Complexes
Type 1 HSV
0.110
0.013
0.136
CMV
? 0.011
(n = 3)
0.125 t 0.011
(n = 3 )
5 0.011
(n = 5 )
0.012 ? 0.013
(n = 5 )
2 0.115'
(n = 6 )
0.141 t 0.066'
(n = 2)
"All values are expressed as mean optical density readings k 1 standard deviation, read at 488 nm.
'Commercial viral antigen was diluted 1: 25 and run in the assay as a positive control.
'Significantly different from negative controls (p < 0.01).
Abbreviations as in Table 1
antibodies reactive with bovine brain galactocerebroside and ganglioside. In addition, 3 subjects in the nonMS group (with SSPE, herpes encephalitis, and partial
complex seizure disorder) showed IC containing galactocerebroside and ganglioside-reactive antibody.
Discussion
ICs are a frequent finding in human diseases, but their
direct relevance is rarely clear. The isolation and characterization of these complexes offer the opportunity
to assess their true role in disease production. Important
information about pathogenesis and causation can be
revealed, particularly with diseases involving unknown
infectious agents or unknown autoimmune targets.
Our initial studies were directed at isolating complexes from a series of disorders involving a known
virus, rubella. We were successful in developing methodology that showed viral-specific complexes in cases
of progressive rubella panencephalitis {5} and congenital rubella infection, and in rubella vaccinees [GI.
The same approach has now been applied to the study
of ICs in patients with MS. Our first goal was to look
for evidence of specific virus involvement. We chose
four ubiquitous agents that are known to cause latent
and neurotropic infections. The second goal was to
look for brain-reactive components, in the hope of
identifying an MS target antigen or significant autoantibody. In this preliminary study, we chose to investigate MBP, galactocerebroside, and ganglioside. All
three are central nervous system antigens that have
been used to produce model autoimmune animal diseases likened to MS {2, 16, 241; MS lymphocytes have
been reported as sensitized to all three {9, 121, and
antibodies against these antigens have been found in
the serum or cerebrospinal fluid of patients with MS
13, 20, 251.
A number of interesting findings resulted from this
initial study. More than 75% of the patients with MS
had serum ICs containing antibodies against at least
one of the four viruses tested. By itself, the finding of a
specific antibody does not necessarily imply the presence of the corresponding antigen, because complexes
can be idiotype-anti-idiotype in nature. Sensitive assays using monoclonal antibody, however, detected
HSVl and CMV viral antigens in at least some of the
MS complexes. These findings suggest that ongoing
systemic viral production is a feature of MS, with reactivation of latent agents perhaps at very low levels. Our
patients were not clinically ill. Moreover, many of
them had circulating complexes involving more than
one virus. Several had viral complexes without corresponding free antibody, as might be expected in a pri-
664 Annals of Neurology Vol 16 No 6 December 1984
Table 4. Clinical Profile of Patients with Mult$le Sclerosis with Viral-Specific lmmune Complexes
Patient
IC
Age (yr),
MS Type, Duration,
Activity, Kurtzke
No.
Level"
Sex
Scoreb
Ab
At:
68
72
71
81
55
50
33
80
40
146
134
55
95
37
38, M
37, M
40, M
54, F
34, F
32, F
51, M
58, M
42, F
37, F
29, M
58, M
42, M
30, M
32, F
38, M
34, F
34, M
32, M
42, M
36, M
ER, 4% yr, stable, 4/1
CP, 17 yr, stable, 23/9
ER, 3 yr, active, 9/12
ER-CP, 12 yr, active, 15/6
CP, 4 yr, active, 1313
CP, 3 yr, active, 1113
CP, 5 yr, active, 1416
ER-CP, 7 yr, active, 912
ER-CP, 7 yr, active, 1214
ER, 3 mo, active, 3/1
ER, 4 yr, active, 812
ER, 7 yr, stable, 2/1
ER, 3 yr, active, 10/3
CP, 4 ?4yr, stable, 11/3
CP, 2% yr, stable, 1013
CP, 5 yr, active, 10/3
ER, 10 yr, active, 12/3
CP, 4 yr, stable, 11/6
CP, 5 yr, stable, 14/5
ER, 21 yr, stable, 9/2
ER, 2 yr, active, 10/5
+
+
+
+
+
+
-
...
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
32
105
102
48
47
20
21
48
32
HSV
+
-
+
+
+
+
+
+
-
+
+
-
-
CMV
Ab
Ag
Measles
Ab
Rubella
Ab
-
+
...
+
+
-
+
-
...
-
...
...
...
...
...
"Values are expressed in micrograms per milliliter of agregated human globulin equivalent.
bStable disease activity indicates no clinical changes in the prior 6 months; active disease activity indicates acute attack in the previous month or
slowly progressive worsening of the prior 6 months. Kurtzke score is expressed as a numerical functionaildisability score.
IC = immune complex; Ab
abbreviations as in Table 1.
=
antibody; Ag = antigen; M = male; F = female; ER = exacerbatinglremitring; CP = chronic progressive; other
Table 5 . lsokzted lmmune Complexes:
BruinSpecific Immune Complexes
MBP
Group
Ab
Ag
Galactocerebroside Ab
Ganglioside
Ab
Controls
(n = 6)
Patients with
non-MS
neurological
disease
(n = 17)
Patients with
MS
(n = 21)
0
...
0
0
2"
...
(12%)
7
5
(33%)
(24%)
3
3
(18%)
(18%)
12
(57%)
(57%)
12
"Included patients with herpes encephalitis and Guillain-Bard syndrome.
MBP = myelin basic protein; Ab = antibody; Ag
sclerosis.
=
antigen; MS = multiple
mary infection. No complexed IgM viral antibody was
found, however. An alternate explanation is that the
available free antibody was bound by the sudden production of virus at a given time. These initial results
raise intriguing questions. Is continuous reactivation of
latent viruses a fundamental abnormality of MS? Could
similar reactivation followed by inflammation be taking
place within brain, with subsequent pathological damage mediated through a bystander effect or a targeted
attack on a cross-reactive viral-brain antigen?
The present study confirmed previous findings that
in patients with MS, brain antigens are ''leaked'' systematically [4, 211. Only one-third of the MS group
showed MBP-specific complexes, however, and we
could detect antigens in only 24%, somewhat less than
the 50% figure reported in a recent study [7]. One
might expect to find an even higher positivity rate if
MBP were the principal target of attack in MS. The
only non-MS subjects who had MBP-specific complexes were a patient with herpes encephalitis and one
with Guillain-Barre syndrome; MBP has been reCoyle and Procyk-Dougherty: MS Immune Complexes 665
ported in both disorders. More than half of the MS
group had complexed antibody reactive with commercial preparations of bovine brain galactocerebroside
and ganglioside. These results are consistent with those
of previous workers; Jacque and colleagues 1131 detected galactocerebroside along with other lipids in
serum complexes of patients with MS. More recently,
Lund and co-workers [17] found that some MS ICs
contained a myelin-membrane-related lipid antigen
that was not galactocerebroside. We found that 3 patients with ONDs with structural brain damage also
had galactocerebroside- and ganglioside-reactive complexes, suggesting that such findings are not MS
specific. Future studies must use very specific and pure
antigen probes and should be expanded to look at
other brain components, including myelin-associated
glycoprotein and proteolipid.
The significance and nature of the IgM and IgA
complexes found in over 60% of the patients with MS,
including 24% with very high IgA levels, are not
known. The presence of IgM suggests novel exposure
to an antigen; IgA suggests antigen entry through mucous membrane. No data are available concerning
whether IgM and IgA complexes are also present in
the cerebrospinal fluid of patients with MS, a factor
that may be important in determining their true relevance.
This preliminary study of serum ICs suggests that
there is continuous reactivation of latent and neurotropic viral agents in patients with MS. Low levels of
virus appear to be produced that are sufficient to form
detectable circulating ICs in the blood but are not
sufficient to produce a clinical disease. Future studies
must be directed at examining cerebrospinal fluid complexes in MS, as well as at determining whether this
viral reactivation is a primary or secondary defect of
affected patients.
Supported by grants from the National Multiple Sclerosis Society
(RG1360A2) and the Kroc Foundation. Dr. Coyle is the recipient of
Teacher-Investigator Development Award NS-00790 from the National Institute of Neurological and Communicative Disorders and
Stroke.
Presented in part of the 108th Annual Meeting of the American
Neurological Association, New Orleans, LA, Oct 1983.
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