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Antibodies to lymphocytes and smooth muscle in the sera of patients with multiple sclerosis
CLINICAL.
IMMUNOLOGY
.4ND
16, 374&38.5
IMMUNOPATHOLOGI
(1980)
Antibodies to Lymphocytes and Smooth Muscle the Sera of Patients with Multiple Sclerosis S.A. MCMILLAN,*
AND D. MIDDLETON+
MARCARETHAIRE,*+
Received
November
in
20. 1979
A higher incidence of cold-reacting lymphocytotoxic antibodies (LC‘A) was found III sera of patients with multiple sclerosis (MS) (5126) compared with healthy controls (1126). When the same sera were tested by indirect immunofluorescence, using as antigen airdried lymphocytes from the patients and controls, antibodies reactive with the cell membranes were found in IgM class more frequently in the MS sera (7126) than in the control sera (l/26). The antibodies were more active against the MS lymphocytes (8126) than against the control cells (4126). Specificity for smooth-muscle-type antigens was found in the antilymphocyte antibodies (ALA), but their presence did not correlate with LCA Evidence for an association of IgM ALA with a rheumatoid-like factor was not found.
INTRODUCTION
Serum antibodies reacting with human lymphocytes have been demonstrated using complement-dependent cytotoxicity techniques and indirect immunofluorescence. Kuwert and Bertrams (1) found an increase in the incidence of coldreacting lymphocytotoxic antibodies (LCA) in the sera of patients in the chronic stages of multiple sclerosis (MS) compared with patients with neurological disorders other than MS and healthy control subjects, when tested against homologous lymphocytes. These LCA appeared to be unrelated to HLA cytotoxic antibody and were probably IgM in character. Schocket rt nl. (2) confirmed these results when they found LCA in the sera of approximately half of their patients with MS compared with low numbers found in the sera of patients with neurological diseases and normal subjects. The specificity of these cold-reacting antibodies has not been determined although it has been suggested that LCA present in the sera of patients with SLE react with the i antigen (3). also found on fetal and neonatal red blood cells. Antilymphocyte antibodies (ALA) detected by indirect immunofluorescence. using as antigen human peripheral lymphocytes fixed in acetone, have been described in several immunoglobulin classes of sera of patients with systemic lupus erythematosus (SLE) (4, 5), and inflammatory bowel disease (IBD) (6), and they have been shown to react with antigen at the lymphocyte surface. Since a higher incidence of smooth-muscle antibody (SMA) in IgM class has been described in the sera of MS patients compared with healthy control subjects (7, 8), and a reaction between sera containing SMA and the surface of peripheral blood lymphocytes has been reported (9), we thought it worthwhile to examine 374
0090-1229/80/070374-12$01.00/O Copynght t$ 1980 by Academic Presc. Inc All rights of reproduction in any form resened
AUTOANTIBODIES
IN
MULTIPLE
SCLEROSIS
375
sera from MS patients and controls for LCA, ALA, and SMA and to compare the prevalence and relationship of antibodies detected. MATERIALS
AND METHODS
Patients and Clinical Specimens Clotted and titrated blood samples were obtained from 26 patients with MS, all probable cases in an inactive phase (lo), and from 26 healthy individuals matched for age and sex. Prior to testing, the sera were stored at -70°C. Lymphocytes, obtained by differential density centrifugation of the titrated blood samples, were suspended in autologous serum at 4 x lo5 cells/ml and cooled at +4”C. They were then stored in l-ml aliquots in liquid nitrogen using an equal volume of 20% dimethyl sulfoxide (BDH Chemicals Ltd., Poole, England) in Hanks’ balanced salt solution (Wellcome Reagents Ltd., Beckenham, England), previously cooled to +4”C, as a cryopreservative. All tests were carried out under code. Sera from three patients with SLE and from two patients with rheumatoid arthritis (RA) were tested in control experiments. Detection of Cold-Reactive Lymphocytotoxic Antibodies LCA were detected using a modified microlymphocytotoxicity technique (1). The target lymphocyte suspensions were obtained from 10 donors, selected to contain the following HLA antigens: HLA -Al, A2, A3, Aw23, Aw24, AlO, Al 1, A28, A29, Aw30, Aw32, HLA -B5, B7, B8, B12, B13, B14, B15, B16, B17, B18, B21, B22, B27, B35, B37. One microliter of serum, inactivated by heat at 56°C for 30 min, was incubated at 15°C for 30 min with 1 ~1 of target lymphocyte suspension (at a concentration of 2.5 x 106 cells/ml), followed by a 3-hr period of incubation at the same temperature after the addition of 5 ~1 of rabbit serum as a source of complement. The sera from three patients with SLE, known to contain LCA, were used as positive control sera. The presence of LCA in each of the 26 MS and 26 control sera was expressed as a cytotoxic index as described by Schocket et al. (2). Cytotoxic
average percentage of target cells killed by test serum index (CI) = x loo. average percentage of target cells killed by the three positive control sera
The presence of HLA cytotoxic antibodies was determined in the same sera, using a two-stage microlymphocytotoxicity test with incubation at 22°C (12). Zmmunojluorescence
Technique
Conjugates. Fluorescein isothiocyanate (FITC)-labeled sheep anti-human IgM (Wellcome Reagents Ltd., Batch No. K5412) and FITC-labeled sheep anti-human IgG (Wellcome Reagents Ltd., Batch No. K7047) were used at their working dilutions, which were assessed by means of a chessboard titration using high- and low-titer IgM and IgG autoantibody-positive sera obtained from patients with various autoimmune conditions.
376
MCMILLAN,
HAIRE,
AND
MIDDLETON
The conjugates were absorbed with acetone-treated rat and mouse liver powders, homogenates of HEp, and BHK cells, and homogenates of rat stomach, kidney, and brain. Detection of serum reactivity with air-dried lymphocytes by indirect immunojkorescence. The method used to test the effect of the sera from patients and control subjects on lymphocyte preparations from each of the 52 individuals was similar to that employed by Husby and Messner (4) with certain modifications. The stored lymphocytes were quickly thawed, and after three washes in 0.01 M phosphate-buffered saline (PBS) were suspended at a concentration of 1.5 x 10fi cells/ml. Ten microliters of the cell suspension was placed on each hole of a Teflon-coated slide, and after rapid drying for 30 min the slides were stored at -20°C until used. The slides were washed in PBS for 30 min and sera were applied to the lymphocytes at a dilution of l/l0 for 30 min at room temperature, and after a further period of washing in PBS for 30 min, anti-human IgM labeled with ffuorescein isothiocyanate was applied for 30 min also at room temperature. The slides were then washed in four changes of PBS for 2 hr to ensure removal of the conjugate. Ethidium bromide (0.5 pg/ml) (Calbiochem Ltd., Hertfordshire, England), which stains nuclei, was applied for 2 set to simplify the identification of nonfluorescing lymphocytes. Finally, after a period of washing in PBS for 5 min, the slides were mounted in glycerol-saline and viewed using an incident light fluorescent microscope. A positive staining reaction was recorded when a serum gave + + or greater staining intensity of 30% or more of lymphocytes in a lymphocyte preparation, and a serum was scored as having antilymphocyte activity only if more than 26 of the 52 lymphocyte preparations (i.e., >50%) showed this positive staining reaction. Since the number of antigen preparations was limited, four MS and four control preparations, which showed reactivity with sera in IgM class, were used to test all sera for activity in IgG class. Sera with antilymphocyte activity (ALA) in both IgM and IgG were titrated on these eight cell preparations, and the percentage of cells stained was determined at each dilution. Detection of Surface lmmunoglobulin Staining c‘elis The effect of anti-human IgM and IgG conjugates alone was tested on each of the 52 lymphocyte preparations and the percentage of fluorescing cells was determined by counting 400 cells. Detection
of Smooth-Muscle
Antibody
Indirect immunofluorescence was used classes in the sera from both patients and composite tissue and acetone-fixed HEp, smooth-muscle antigen of similar specificity
to detect antibodies in IgM and IgG control subjects using as antigen rat cells, shown previously to contain (13).
Absorption of Sera Three MS and two control sera, containing parallel at a dilution of l/5 with the following
IgM antibodies were absorbed in absorbants: human myometrium,
AUTOANTIBODIES
IN
MULTlPLE
SCLEROSIS
377
normal brain, MS brain, and a packed cell suspension of cord red blood cells (RBC) and RBC of blood groups AB, A, B, and 0 (final concentration 20% v/v). After a period of 48-hr rotation at +4”C the sera were centrifuged and clarified by filtration. Dilutions of the absorbed and unabsorbed sera were tested in parallel and the titer of antibody was estimated. The antigenic specificity of IgM smoothmuscle-type antibody (SMA) in two of these MS sera had been previously determined by testing the sera on tissue culture cells treated with certain cytotoxic drugs (14). Determincttion
of Rheumatoid
Factor (RF)
The Hyland RA rapid slide test was used to determine the presence and titer of RF in the 52 sera of the study (Travenol Laboratories, Costa Mesa, Calif.). Eight sera from MS patients with ALA and SMA in IgM class and two sera from RA patients with high levels of RF were absorbed twice at a serum dilution of l/10 for 1 hr at room temperature with a 20% (v/v) of a packed suspension of washed Hyland latex coated with y-globulin (Travenol Laboratories). After centrifugation and filtration the sera were tested in parallel with the unabsorbed sera for the presence of RF, SMA, and ALA in IgM class. Serum Fractionation
Experiments
Fractions containing IgG and IgM were obtained by column chromatography (15) from sera from three patients with MS, which showed ALA in IgM class. Similar fractions were also obtained from one control serum with, and two control sera without, ALA. IgG- and similar IgM-containing fractions were pooled, and the monospecificity of each pool was ensured by testing each by gel diffusion and immunoelectrophoresis against monospecific antisera. Dilutions of the sera and the corresponding IgG and IgM serum fractions were tested for ALA in both IgG and IgM classes, using as antigen one control and two MS lymphocyte preparations. RESULTS
Incidence of Cold-Reactive Lymphocytotoxic Antibodies When a CI value of 15 was used, 7/26 MS sera (27%) contained LCA compared with 4/26 control sera (lS%), a nonsignificant difference (0.1 > P > 0.05). With a CI value of 40,5/26 MS sera (20%) compared with l/26 control sera (4%) contained LCA and while the difference between the groups was slightly greater, it was not of statistical significance (0.5 > P > 0.3) (Fig. 1). These 5 MS sera were later used for correlation studies of different serum reactivities. HLA cytotoxic antibodies were only detected in 2 MS sera which did not contain any LCA. Incidence
of Surface Immunoglobulin
Staining
Cells
A low percentage of lymphocytes from each group showed surface staining with both the anti-human IgG and anti-human IgM conjugates (Table l), probably due to surface immunoglobulin present on B lymphocytes. These cells were included in the cell count in the indirect immunofluorescent test for the detection of serum reactivity with lymphocytes.
378
MCMILLAN,
HAIRE,
AND
MIDDLETON
u 3-
60
.o
:
2
50
”
40
0 x
l
-2,
---
30
is -e-e 0
%i@c 00 control
MS FIG. 1. The distribution and 40 are indicated.
Imklencr
$
sera sera of cytotoxic indices in the 26 MS and 26 control sera. The Cl values of 15
of Serurn Reactivity
Mith Air-Dried
Lgmphocytt~s
(ALA )
Reactivity with membrane antigen(s) of air-dried lymphocytes was detected in both the MS and control groups of sera by indirect immunofluorescence. The staining pattern was observed as a rim or ring on the periphery of each lymphoTABLE THE AVERAGE
INCIDENCE OF LYMPHOCYTES WITH Two AWI-HUMAN IMMUNOGLOBULIN PATIENTS
Immunoglobulin class
Number in group
AND
___-
THE
26
I SURFACE
IMMLJNOGLOBULINS
CONJUGATES NORMAL.
IN THF ?6
DETECTELI
BY
MS
SUBJECTS
Average percentage of cells ” with surface immunoglobulins in the lymphocyte preparations From MS group
From control group
W
0.7 If 2.1
I&f
5.25 i- 1.8
0.9 z 1.9 4.25 z 2.4
26
26
” 400 cells counted in each patient’s lymphocyte preparation
rHE
AUTOANTIBODIES
IN MULTIPLE
SCLEROSIS
379
cyte, and combination of immunofluorescence and phase microscopy indicated that the staining was at the cell surface and not in the nucleus. All sera which were considered negative gave weak fluorescent staining of all lymphocyte preparations used, whereas this staining was not present in the control preparations to which PBS had been applied prior to incubation with the conjugates. There was a significant difference (0.05 > P > 0.02) in the incidence of activity in IgM class detected by both MS and control lymphocyte preparations in the sera of the MS patients (7/26) compared with those in the control sera (l/26). However, the number of MS and control sera considered positive differed when either the MS or control lymphocytes were used as antigen (Fig. 2, Table 2). Eight of the 26 MS sera were considered positive when the MS cells were used as antigen whereas only 4 of these were positive when the control cells were used, and only two sera stained 100% of both the MS and control cells. Similarly the positive sera from the control group showed more reactivity with the MS cells than with the control cells. As well as this difference in the reactivity of the sera seen when either the MS or control lymphocytes were used as antigen, each of the positive sera also showed a variation in the number of lymphocytes which they stained in each of the lympho-
MS C&S (n. 26) 1oc
IT
90
I_
.-m z
80
g .-
70
[
60
(n=26)
MS cells
Cglll~’
(t-t= 26)
(n=26)
t
MS sera Control sera 2. The percentage of either MS or control lymphocyte preparations stained by ALA in the MS and control sera. ’ Sera giving a + + or + + + immunofluorescent intensity of more than 30% of each target lymphocyte preparations. FIG.
380
MCMILLAN,
HAIRE, AND MIDIXETON
TABLE 2 THE IWCIDENC~ OF ALA IN 1gM CLASS PREsEN,r I& I HF Two LYMPHOCYTES FROM BOTH MS PATIENTS END
SLKLV GROLIPS tkmc CON GROIN S~JBJECTS
11.1) HI
Number of sera containing ALA” Target lymphocytes used
7 x 4
1‘, 1 (I :
26
26
MS and control (II = 52) MS (II = 26) Control (II = 26) Number of sera tested ” Positivity was I, x2 (I) = 4.18. I’ x” (1) = 5.42. ” x? (1) = 2.25.
In control group
In MS group
taken as staining of 50% or more of the target lymphocyte preparations 0.05 Z P > 0.02, significant. 0.05 1 P > 0.02, significant. 0.2 > P :’ 0.1. not significant.
cyte preparations, and the average of this variation seen in the positive MS sera ranged from 46 to 96%. The titer of this activity present in the 7 MS sera ranged between Ii20 and l/80 with the majority (5/7) containing activity at titer 1140, while one control serum had activity to a titer of l/40. Two out of twenty-six MS sera (titers l/20 and 1140’1 and 1126 control sera (titer l/20) contained ALA in IgG class, and only one of these 2 MS sera contained a corresponding ALA in IgM class. The one control serum contained ALA in both IgM and IgG classes. incidence
of’ Smooth-Muscle
Antibody
An increased incidence of SMA in IgM class detected on rat composite tissue was found in the MS group (9/26) compared with the control group (4/26), but the difference was not of statistical significance (0.2 1 P > 0.1) in the present study. There was a significantly higher incidence (0.02 1 P B 0.01) of IgM class SMA detected on HEp, cells in the MS group (B/26) compared with the control group (2/26) (Table 3). These antibodies were infrequent in IgG class. TABLE THE
INCIDEKCE
OF
SMA
IN IN SERA
IgM
AND
OF
MS
3
IgG CLASSES PATIEUTS .AND
DFJE~‘IE~ hr TIMER 01. 1120 OR GKF:*F~K COWKOL SURJECTS l__Incidence of antibody
In MS serum Antigen Rat composite tissue HEp, tissue culture cells
Wf
I& ---~-..~..~.----..~ 1 0
9’1 8”
Number of sera tested (’ ,yz(1) = 2.28, 0.2 > P > 0.1, not significant. ” xL (1) = 6.125. 0.02 3) P > 0.01, significant.
26
In control serum k&J 4” 2”
IgG ~~- ~~.~-~~ ~. 2 0
26
AUTOANTIBODIES
IN
MULTIPLE
381
SCLEROSIS
Comparisorz of the Lymphocytotoxic Antibodies (LCA) in the Sera of the MS Patients with Antibodies to Lymphocytes and Smooth Muscle in IgM Class A negative correlation was found between the occurrence in the sera of LCA and antilymphocyte activity in IgM class (Table 4). A similar negative correlation was also found between the occurrence of LCA and SMA detected by rat composite tissue and HEp, tissue culture cells. However, a positive correlation was found between the occurrence of antilymphocyte activity and SMA. The titers in the same serum were within a twofold dilution of each other, with the exception that serum 17 17 contained antibody to smooth muscle (l/160 detected by rat tissue, l/80 detected by HEp, cells) but did not react with the air-dried lymphocytes. In contrast serum 1694, while containing ALA, did not contain SMA. The Speci’city of the IgM Anticellular Antibodies Absorption of the sera with human myometrium (uterus), MS brain, and normal brain caused a reduction in titer of both the SMA and ALA in IgM class, whereas TABLE 4 COMPARISON OF LCA, ALA, AND SMA IN SERA OF 26 MS PATIENTS Type of antibody Serum
LCA
1718 1747 1684 1707 1685 1692 1695 1673 1717 1749 1691 1779 1694
+ +
Total positive
40
40
+ + + 5 LCA+
SMA” (Rat tissue)
ALA”
80 40
40 40 110
9
8
7 =CI>40
LCA-
Negative correlation between LCA and ALA LCA and SMA (rat tissue) LCA and SMA (HEp, cells) Positive correlation between ALA and SMA (rat tissue) SMA (rat tissue) and SMA (HEp, cells) ALA and SMA (HEp, cells) ” These are in IgM class. Note: Antibody titers are expressed as reciprocals.
SMA” (HEp, cells)
=CI<40 P = 0.5875 P = 1.000 P = 1.000 P = 0.0022
P = 0.0019 P = 0.0019
(Cl (‘2)
(MS)
(MS) (MS)
____-
40 80 160 20 40
SMA titer
OF SMA
10
IO
IO
10 40 40
10
IO 20 20
MS brain
10
10
10 40 40
Normal brain
CLASS OF MS
SMA after with
oh THE IgM
of IgM absorption
Uterus
-____
Titer
&ND ALA
40 - ___-.-.
20
40 80 160
RBC pool
TABLE ASD CONTROL
40 40 NT’ 40 NT
ALA titer
5 SER.\
_.._
(91 i
(100) (93)
(‘,;; ) i,
_
Titer
~EFORF
XFTFR
ABSORPII~K
-
10
10 (84)
10
~-.Normal brain
VARIOC;~
preparationa.
- .___ -_ ----.--lymphocyte
_---.
I0
IO (97)
20 190) IO (9.5)
10
MS brain
- --
MIT-H
10
Uterus
of IgM ALA” after absorption with -__.--~. ~-
\h;~
___II_-. “1’0~c. Antibody titers are expressed as reciprocal\. ’ The titers of the ‘41~4 in the IgM cl;\\\ were average< uf rhtlsz detected on IWU MS and two control ” Average percentage of sells giving po\itivc \taining (i.e.. or - I OII the four cell prepurati0n.r. ’ NT. not tested.
1747 1718 1717 2007 2004
Serum
THY TIIERS
.--
-
40 (89,
40 (100) 40 (961
RBC pool
. -.-----
- --
ABSORIL+NIS
: 3 E; 0 L
2 ;
T
5
5
5
w w
AUTOANTIBODIES
IN MULTIPLE
383
SCLEROSIS
absorption with human rbc had no effect (Table 5). The ability of the myometrium, normal and MS brain appeared to be somewhat similar in absorbing out IgM activity. RF was detected in 1126 MS sera, and absorption with latex-coated y-globulin removed the low-titer RF from the serum but only slightly reduced the titers of both SMA and ALA in IgM class from the sera of the eight patients with MS (Table 6). Similar absorption of 2 sera from patients with RA with high-titer RF, reduced the titer eightfold. The ALA activity detected in either IgG or IgM class of the whole serum by indirect immunofluorescence was also found in the corresponding immunoglobulin class fractions, when tests were carried out on sera from three patients with MS and from a healthy person (Table 7). DISCUSSION
We have not found so high an incidence of LCA in sera of MS patients as previous workers (2, 16), but the lack of correlation we have shown between LCA and HLA cytotoxic antibodies is similar to that shown by Kuwert and Bertrams (1). We have found a significantly higher level of ALA in IgM class in MS compared with control sera, and the staining pattern in the immunofluorescence test appears to be similar to the description of Husby and Messner (4) and Searles, Husby, and Messner (5) of the staining of lymphocytes by SLE sera. Antilymphocyte antibodies (ALA) have not been correlated with LCA in the sera of SLE patients (17), of relatives of SLE patients (5), and of patients with IBD (6). Similarly our results did not show any correlation between LCA and ALA. However, we have shown a correlation between ALA and SMA in IgM, and this may point to the occurrence of antigen or antigens of similar specificity in the
THE EFFECT
TABLE 6 OF MS AND RA SERA WITH LATEX ON VARIOUS ANTIBODY TITERS
OF ABSORPTION GLOBULIN
Titer of antibodies” In serum absorbed with latex y-globulin
In unabsorbed serum Sera 1718 (MS) 1695 (MS) 1747 (MS) 1673 (MS) 1694 (MS) 1779 (MS) 1692 (MS) 2230 (MS) 9158 (RA) 9064 (RA)
RF
SMA
ALA (‘%)b
RF
20 20 20 20
40 (78) 20 (63) 20 (91) 10 (52) 20 (83) 20 (58) 40 (96) 40 (94)
SMA 20 10 20 10
ALA (%)” 40 (75) 20 (68) 10 (81) 10 (61) 10 (87) 10 (43) 20 (98) 40 (91) Cl0
Note. Antibody titers are expressed as reciprocals. ” The SMA, and ALA were all in IgM class. I’ Mean percentage of cells staining using one MS and one normal lymphocyte preparation.
384
MCMILLAN,
HAIRE,
TABLE THE
PRESENCE
OF IgG
AND
IgM
ALA
IN WHOLE
AND
MIDDLETON
7 SERUM
AND
IgG
AND
IgM SERUM Faxrroxs
Presence of ALA staining” -.-~ In whole serum at dilution 1120 Serum 1747 (MS) 1692 (MS) 1718 (MS) 2007 CC) 1974 (0 I973 (f.3
G
M
In IgG fraction
In
G
G
M
IgM
fracrion M
-~ +
t
^
+ -
+
^
_.
‘I The results were obtained in duplicate using a normal lymphocyte preparation as antigen.
three antigens used, i.e., lymphocytes, rat composite tissue, and cultured HEp, cells, and absorption studies using human uterus, normal, and MS brain suggest a smooth-muscle antigen specificity. In two of these MS sera, the specificity of the SMA in IgM has been shown to be against a component of cell intermediate filaments (14). Since absorption of sera with pooled-adult and cord rbc did not affect the titers of SMA it is indicated that antibody was not directed against the antigens of the Ii system. The finding that MS lymphocytes were a more sensitive antigen than control cells to detect ALA may indicate an increase of smooth-muscle-type antigen made available in lymphocytes by viral or other stimulation, somewhat similar to the enhancing effect of phytohemagglutinin (PHA) on smooth-muscle antigen in peripheral blood lymphocytes (9). Since RF in serum has been shown to be responsible for false positive IgM staining of viral antibodies in the immunofluorescent technique (18), and IgM staining of measles virus in the sera of MS patients has been described as being caused by a rheumatoid-like factor (19, 20), experiments were carried out to ensure that RF was not responsible for the staining patterns found when testing for ALA and SMA in IgM class. False positive viral IgM staining is only found in the presence of the corresponding viral antibody in IgG class (18, 21). ALA in IgG class was infrequent and indeed ALA in IgM class was shown in the absence of ALA in IgG class. In addition, our results show that ALA was not removed by absorption with latex-coated y-globulin, and ALA in IgM class was only detected in IgM fractions of sera. The slightly higher incidence of ALA and SMA in IgM class in MS patients compared with controls may result from alteration in T-cell activity. A persistent viral infection may cause a loss of suppressor T-cell function or may cause a loss of tolerance to autoantigens of helper T cells. The reaction of ALA with membranes of lymphocytes, with increased activity with patients’ cells, may indicate an alteration of receptors on lymphocytes for IgM in MS. Since the patients in this study were all probable cases in an inactive phase, a further study is warranted of autoantibodies to smooth muscle and subpopulations of lymphocyte membranes in MS patients with active disease.
AUTOANTIBODIES
IN MULTIPLE
SCLEROSIS
385
ACKNOWLEDGMENTS This work has been made possible by the collaboration of many colleagues. The patients were under the care of Dr. J. H. D. Millar, who also read the manuscript. Colonel T. D. Field made available the normal control specimens. Dr. J. D. Merrett, Department of Medical Statistics, The Queen’s University of Belfast, carried out the statistical analyses. Dr. P. V. Shirodaria gave valuable criticism and advice. Mrs. E. E. Russell, Mrs. J. P. Douglas, Mrs. J. Totton, Mrs. C. Sleator, and Mrs. S. A. Kirk provided technical assistance. Miss J. Smyth typed the manuscript. We wish to thank these colleagues. Part of the work carried out in the Department of Microbiology and Immunobiology, The Queen’s University of Belfast, is supported by a Program Grant from the Medical Research Council.
REFERENCES 1. Kuwert, E., and Bertrams, J.. Eur. Neural. 7, 65, 1972. 2. Schocket, A. L., Weiner, H. L., Walker, J., McIntosh, K., and Kohler. P. F., C/in. fmmunol. Immunopatld.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
7, 15, 1977.
Goldberg, L. S., Bresnihan, B., and Hughes, G. R. V., C/in. Exp. fmmunol. 31, 443, 1978. Husby, G., and Messner, R. P.. J. Lab. Clin. Med. 89, 240, 1977. Searles, R. P.. Husby. G., and Messner, R. P., Acta Pathol. Microbial. Scund. 74C, 463, 1977. Henderson, C. A., Greenlee, L., Williams, R. C.. Jr., and Strickland, R. G., Scund. J. Immunol. 5, 837, 1976. Haire, M., &it. Med. Bull. 33, 40, 1977. McMillan, S. A., and Haire, M., unpublished data. Fagraeus, A., Lidman, K., and Biberfeld, G., Nature (London) 252, 246, 1974. Millar, J. H. D., Fraser, K. B.. Haire, M., Connolly, J. H., Shirodaria, P. V., and Hadden, D. S. M., h-it. Med. J. 2, 378, 1971. Mottironi, V. D., and Terasaki, P. I., In “Histocompatibility Testing” (P. I. Terasaki, Ed.), pp. 301-308, Munksgaard, Copenhagen, 1970. Nelson, S. D., and Middleton, D., Ulster Med. J. 44, 48, 1975. McMillan, S. A., and Haire, M.. C/in. Exp. Immunol. 21, 339, 1975. McMillan, S. A., and Haire, M., C/in. Immunol. Immunopathol. 14, 256, 1979. Hannon. R., Haire. M., Wisdom. G. B.. and Neil], D. W., J. Immunol. Methods 8, 29, 1975. Schocket. A. L., and Weiner, H. L., Lancer 1, 571, 1978. Messner, R. P., Kennedy, M. S., and Jelinek, J. G., Arthritis Rheum. 18, 201, 1975. Shirodaria. P. V.. Fraser. K. B., and Stanford, F., Ann. Rherrm. Dis. 32. 53, 1973. Fraser, K. B., Lancrt 1, 91, 1978. Fraser, K. B., Armstrong, M.. Shirodaria. P. V., and Gharpure, M., C/in. Exp. Immunol. 36, 304, 1979. Shirodaria, P. V., Fraser, K. B., Armstrong. M.. and Roberts, S. D., Infer. Immunity 25, 408, 1979.
IMMUNOLOGY
.4ND
16, 374&38.5
IMMUNOPATHOLOGI
(1980)
Antibodies to Lymphocytes and Smooth Muscle the Sera of Patients with Multiple Sclerosis S.A. MCMILLAN,*
AND D. MIDDLETON+
MARCARETHAIRE,*+
Received
November
in
20. 1979
A higher incidence of cold-reacting lymphocytotoxic antibodies (LC‘A) was found III sera of patients with multiple sclerosis (MS) (5126) compared with healthy controls (1126). When the same sera were tested by indirect immunofluorescence, using as antigen airdried lymphocytes from the patients and controls, antibodies reactive with the cell membranes were found in IgM class more frequently in the MS sera (7126) than in the control sera (l/26). The antibodies were more active against the MS lymphocytes (8126) than against the control cells (4126). Specificity for smooth-muscle-type antigens was found in the antilymphocyte antibodies (ALA), but their presence did not correlate with LCA Evidence for an association of IgM ALA with a rheumatoid-like factor was not found.
INTRODUCTION
Serum antibodies reacting with human lymphocytes have been demonstrated using complement-dependent cytotoxicity techniques and indirect immunofluorescence. Kuwert and Bertrams (1) found an increase in the incidence of coldreacting lymphocytotoxic antibodies (LCA) in the sera of patients in the chronic stages of multiple sclerosis (MS) compared with patients with neurological disorders other than MS and healthy control subjects, when tested against homologous lymphocytes. These LCA appeared to be unrelated to HLA cytotoxic antibody and were probably IgM in character. Schocket rt nl. (2) confirmed these results when they found LCA in the sera of approximately half of their patients with MS compared with low numbers found in the sera of patients with neurological diseases and normal subjects. The specificity of these cold-reacting antibodies has not been determined although it has been suggested that LCA present in the sera of patients with SLE react with the i antigen (3). also found on fetal and neonatal red blood cells. Antilymphocyte antibodies (ALA) detected by indirect immunofluorescence. using as antigen human peripheral lymphocytes fixed in acetone, have been described in several immunoglobulin classes of sera of patients with systemic lupus erythematosus (SLE) (4, 5), and inflammatory bowel disease (IBD) (6), and they have been shown to react with antigen at the lymphocyte surface. Since a higher incidence of smooth-muscle antibody (SMA) in IgM class has been described in the sera of MS patients compared with healthy control subjects (7, 8), and a reaction between sera containing SMA and the surface of peripheral blood lymphocytes has been reported (9), we thought it worthwhile to examine 374
0090-1229/80/070374-12$01.00/O Copynght t$ 1980 by Academic Presc. Inc All rights of reproduction in any form resened
AUTOANTIBODIES
IN
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375
sera from MS patients and controls for LCA, ALA, and SMA and to compare the prevalence and relationship of antibodies detected. MATERIALS
AND METHODS
Patients and Clinical Specimens Clotted and titrated blood samples were obtained from 26 patients with MS, all probable cases in an inactive phase (lo), and from 26 healthy individuals matched for age and sex. Prior to testing, the sera were stored at -70°C. Lymphocytes, obtained by differential density centrifugation of the titrated blood samples, were suspended in autologous serum at 4 x lo5 cells/ml and cooled at +4”C. They were then stored in l-ml aliquots in liquid nitrogen using an equal volume of 20% dimethyl sulfoxide (BDH Chemicals Ltd., Poole, England) in Hanks’ balanced salt solution (Wellcome Reagents Ltd., Beckenham, England), previously cooled to +4”C, as a cryopreservative. All tests were carried out under code. Sera from three patients with SLE and from two patients with rheumatoid arthritis (RA) were tested in control experiments. Detection of Cold-Reactive Lymphocytotoxic Antibodies LCA were detected using a modified microlymphocytotoxicity technique (1). The target lymphocyte suspensions were obtained from 10 donors, selected to contain the following HLA antigens: HLA -Al, A2, A3, Aw23, Aw24, AlO, Al 1, A28, A29, Aw30, Aw32, HLA -B5, B7, B8, B12, B13, B14, B15, B16, B17, B18, B21, B22, B27, B35, B37. One microliter of serum, inactivated by heat at 56°C for 30 min, was incubated at 15°C for 30 min with 1 ~1 of target lymphocyte suspension (at a concentration of 2.5 x 106 cells/ml), followed by a 3-hr period of incubation at the same temperature after the addition of 5 ~1 of rabbit serum as a source of complement. The sera from three patients with SLE, known to contain LCA, were used as positive control sera. The presence of LCA in each of the 26 MS and 26 control sera was expressed as a cytotoxic index as described by Schocket et al. (2). Cytotoxic
average percentage of target cells killed by test serum index (CI) = x loo. average percentage of target cells killed by the three positive control sera
The presence of HLA cytotoxic antibodies was determined in the same sera, using a two-stage microlymphocytotoxicity test with incubation at 22°C (12). Zmmunojluorescence
Technique
Conjugates. Fluorescein isothiocyanate (FITC)-labeled sheep anti-human IgM (Wellcome Reagents Ltd., Batch No. K5412) and FITC-labeled sheep anti-human IgG (Wellcome Reagents Ltd., Batch No. K7047) were used at their working dilutions, which were assessed by means of a chessboard titration using high- and low-titer IgM and IgG autoantibody-positive sera obtained from patients with various autoimmune conditions.
376
MCMILLAN,
HAIRE,
AND
MIDDLETON
The conjugates were absorbed with acetone-treated rat and mouse liver powders, homogenates of HEp, and BHK cells, and homogenates of rat stomach, kidney, and brain. Detection of serum reactivity with air-dried lymphocytes by indirect immunojkorescence. The method used to test the effect of the sera from patients and control subjects on lymphocyte preparations from each of the 52 individuals was similar to that employed by Husby and Messner (4) with certain modifications. The stored lymphocytes were quickly thawed, and after three washes in 0.01 M phosphate-buffered saline (PBS) were suspended at a concentration of 1.5 x 10fi cells/ml. Ten microliters of the cell suspension was placed on each hole of a Teflon-coated slide, and after rapid drying for 30 min the slides were stored at -20°C until used. The slides were washed in PBS for 30 min and sera were applied to the lymphocytes at a dilution of l/l0 for 30 min at room temperature, and after a further period of washing in PBS for 30 min, anti-human IgM labeled with ffuorescein isothiocyanate was applied for 30 min also at room temperature. The slides were then washed in four changes of PBS for 2 hr to ensure removal of the conjugate. Ethidium bromide (0.5 pg/ml) (Calbiochem Ltd., Hertfordshire, England), which stains nuclei, was applied for 2 set to simplify the identification of nonfluorescing lymphocytes. Finally, after a period of washing in PBS for 5 min, the slides were mounted in glycerol-saline and viewed using an incident light fluorescent microscope. A positive staining reaction was recorded when a serum gave + + or greater staining intensity of 30% or more of lymphocytes in a lymphocyte preparation, and a serum was scored as having antilymphocyte activity only if more than 26 of the 52 lymphocyte preparations (i.e., >50%) showed this positive staining reaction. Since the number of antigen preparations was limited, four MS and four control preparations, which showed reactivity with sera in IgM class, were used to test all sera for activity in IgG class. Sera with antilymphocyte activity (ALA) in both IgM and IgG were titrated on these eight cell preparations, and the percentage of cells stained was determined at each dilution. Detection of Surface lmmunoglobulin Staining c‘elis The effect of anti-human IgM and IgG conjugates alone was tested on each of the 52 lymphocyte preparations and the percentage of fluorescing cells was determined by counting 400 cells. Detection
of Smooth-Muscle
Antibody
Indirect immunofluorescence was used classes in the sera from both patients and composite tissue and acetone-fixed HEp, smooth-muscle antigen of similar specificity
to detect antibodies in IgM and IgG control subjects using as antigen rat cells, shown previously to contain (13).
Absorption of Sera Three MS and two control sera, containing parallel at a dilution of l/5 with the following
IgM antibodies were absorbed in absorbants: human myometrium,
AUTOANTIBODIES
IN
MULTlPLE
SCLEROSIS
377
normal brain, MS brain, and a packed cell suspension of cord red blood cells (RBC) and RBC of blood groups AB, A, B, and 0 (final concentration 20% v/v). After a period of 48-hr rotation at +4”C the sera were centrifuged and clarified by filtration. Dilutions of the absorbed and unabsorbed sera were tested in parallel and the titer of antibody was estimated. The antigenic specificity of IgM smoothmuscle-type antibody (SMA) in two of these MS sera had been previously determined by testing the sera on tissue culture cells treated with certain cytotoxic drugs (14). Determincttion
of Rheumatoid
Factor (RF)
The Hyland RA rapid slide test was used to determine the presence and titer of RF in the 52 sera of the study (Travenol Laboratories, Costa Mesa, Calif.). Eight sera from MS patients with ALA and SMA in IgM class and two sera from RA patients with high levels of RF were absorbed twice at a serum dilution of l/10 for 1 hr at room temperature with a 20% (v/v) of a packed suspension of washed Hyland latex coated with y-globulin (Travenol Laboratories). After centrifugation and filtration the sera were tested in parallel with the unabsorbed sera for the presence of RF, SMA, and ALA in IgM class. Serum Fractionation
Experiments
Fractions containing IgG and IgM were obtained by column chromatography (15) from sera from three patients with MS, which showed ALA in IgM class. Similar fractions were also obtained from one control serum with, and two control sera without, ALA. IgG- and similar IgM-containing fractions were pooled, and the monospecificity of each pool was ensured by testing each by gel diffusion and immunoelectrophoresis against monospecific antisera. Dilutions of the sera and the corresponding IgG and IgM serum fractions were tested for ALA in both IgG and IgM classes, using as antigen one control and two MS lymphocyte preparations. RESULTS
Incidence of Cold-Reactive Lymphocytotoxic Antibodies When a CI value of 15 was used, 7/26 MS sera (27%) contained LCA compared with 4/26 control sera (lS%), a nonsignificant difference (0.1 > P > 0.05). With a CI value of 40,5/26 MS sera (20%) compared with l/26 control sera (4%) contained LCA and while the difference between the groups was slightly greater, it was not of statistical significance (0.5 > P > 0.3) (Fig. 1). These 5 MS sera were later used for correlation studies of different serum reactivities. HLA cytotoxic antibodies were only detected in 2 MS sera which did not contain any LCA. Incidence
of Surface Immunoglobulin
Staining
Cells
A low percentage of lymphocytes from each group showed surface staining with both the anti-human IgG and anti-human IgM conjugates (Table l), probably due to surface immunoglobulin present on B lymphocytes. These cells were included in the cell count in the indirect immunofluorescent test for the detection of serum reactivity with lymphocytes.
378
MCMILLAN,
HAIRE,
AND
MIDDLETON
u 3-
60
.o
:
2
50
”
40
0 x
l
-2,
---
30
is -e-e 0
%i@c 00 control
MS FIG. 1. The distribution and 40 are indicated.
Imklencr
$
sera sera of cytotoxic indices in the 26 MS and 26 control sera. The Cl values of 15
of Serurn Reactivity
Mith Air-Dried
Lgmphocytt~s
(ALA )
Reactivity with membrane antigen(s) of air-dried lymphocytes was detected in both the MS and control groups of sera by indirect immunofluorescence. The staining pattern was observed as a rim or ring on the periphery of each lymphoTABLE THE AVERAGE
INCIDENCE OF LYMPHOCYTES WITH Two AWI-HUMAN IMMUNOGLOBULIN PATIENTS
Immunoglobulin class
Number in group
AND
___-
THE
26
I SURFACE
IMMLJNOGLOBULINS
CONJUGATES NORMAL.
IN THF ?6
DETECTELI
BY
MS
SUBJECTS
Average percentage of cells ” with surface immunoglobulins in the lymphocyte preparations From MS group
From control group
W
0.7 If 2.1
I&f
5.25 i- 1.8
0.9 z 1.9 4.25 z 2.4
26
26
” 400 cells counted in each patient’s lymphocyte preparation
rHE
AUTOANTIBODIES
IN MULTIPLE
SCLEROSIS
379
cyte, and combination of immunofluorescence and phase microscopy indicated that the staining was at the cell surface and not in the nucleus. All sera which were considered negative gave weak fluorescent staining of all lymphocyte preparations used, whereas this staining was not present in the control preparations to which PBS had been applied prior to incubation with the conjugates. There was a significant difference (0.05 > P > 0.02) in the incidence of activity in IgM class detected by both MS and control lymphocyte preparations in the sera of the MS patients (7/26) compared with those in the control sera (l/26). However, the number of MS and control sera considered positive differed when either the MS or control lymphocytes were used as antigen (Fig. 2, Table 2). Eight of the 26 MS sera were considered positive when the MS cells were used as antigen whereas only 4 of these were positive when the control cells were used, and only two sera stained 100% of both the MS and control cells. Similarly the positive sera from the control group showed more reactivity with the MS cells than with the control cells. As well as this difference in the reactivity of the sera seen when either the MS or control lymphocytes were used as antigen, each of the positive sera also showed a variation in the number of lymphocytes which they stained in each of the lympho-
MS C&S (n. 26) 1oc
IT
90
I_
.-m z
80
g .-
70
[
60
(n=26)
MS cells
Cglll~’
(t-t= 26)
(n=26)
t
MS sera Control sera 2. The percentage of either MS or control lymphocyte preparations stained by ALA in the MS and control sera. ’ Sera giving a + + or + + + immunofluorescent intensity of more than 30% of each target lymphocyte preparations. FIG.
380
MCMILLAN,
HAIRE, AND MIDIXETON
TABLE 2 THE IWCIDENC~ OF ALA IN 1gM CLASS PREsEN,r I& I HF Two LYMPHOCYTES FROM BOTH MS PATIENTS END
SLKLV GROLIPS tkmc CON GROIN S~JBJECTS
11.1) HI
Number of sera containing ALA” Target lymphocytes used
7 x 4
1‘, 1 (I :
26
26
MS and control (II = 52) MS (II = 26) Control (II = 26) Number of sera tested ” Positivity was I, x2 (I) = 4.18. I’ x” (1) = 5.42. ” x? (1) = 2.25.
In control group
In MS group
taken as staining of 50% or more of the target lymphocyte preparations 0.05 Z P > 0.02, significant. 0.05 1 P > 0.02, significant. 0.2 > P :’ 0.1. not significant.
cyte preparations, and the average of this variation seen in the positive MS sera ranged from 46 to 96%. The titer of this activity present in the 7 MS sera ranged between Ii20 and l/80 with the majority (5/7) containing activity at titer 1140, while one control serum had activity to a titer of l/40. Two out of twenty-six MS sera (titers l/20 and 1140’1 and 1126 control sera (titer l/20) contained ALA in IgG class, and only one of these 2 MS sera contained a corresponding ALA in IgM class. The one control serum contained ALA in both IgM and IgG classes. incidence
of’ Smooth-Muscle
Antibody
An increased incidence of SMA in IgM class detected on rat composite tissue was found in the MS group (9/26) compared with the control group (4/26), but the difference was not of statistical significance (0.2 1 P > 0.1) in the present study. There was a significantly higher incidence (0.02 1 P B 0.01) of IgM class SMA detected on HEp, cells in the MS group (B/26) compared with the control group (2/26) (Table 3). These antibodies were infrequent in IgG class. TABLE THE
INCIDEKCE
OF
SMA
IN IN SERA
IgM
AND
OF
MS
3
IgG CLASSES PATIEUTS .AND
DFJE~‘IE~ hr TIMER 01. 1120 OR GKF:*F~K COWKOL SURJECTS l__Incidence of antibody
In MS serum Antigen Rat composite tissue HEp, tissue culture cells
Wf
I& ---~-..~..~.----..~ 1 0
9’1 8”
Number of sera tested (’ ,yz(1) = 2.28, 0.2 > P > 0.1, not significant. ” xL (1) = 6.125. 0.02 3) P > 0.01, significant.
26
In control serum k&J 4” 2”
IgG ~~- ~~.~-~~ ~. 2 0
26
AUTOANTIBODIES
IN
MULTIPLE
381
SCLEROSIS
Comparisorz of the Lymphocytotoxic Antibodies (LCA) in the Sera of the MS Patients with Antibodies to Lymphocytes and Smooth Muscle in IgM Class A negative correlation was found between the occurrence in the sera of LCA and antilymphocyte activity in IgM class (Table 4). A similar negative correlation was also found between the occurrence of LCA and SMA detected by rat composite tissue and HEp, tissue culture cells. However, a positive correlation was found between the occurrence of antilymphocyte activity and SMA. The titers in the same serum were within a twofold dilution of each other, with the exception that serum 17 17 contained antibody to smooth muscle (l/160 detected by rat tissue, l/80 detected by HEp, cells) but did not react with the air-dried lymphocytes. In contrast serum 1694, while containing ALA, did not contain SMA. The Speci’city of the IgM Anticellular Antibodies Absorption of the sera with human myometrium (uterus), MS brain, and normal brain caused a reduction in titer of both the SMA and ALA in IgM class, whereas TABLE 4 COMPARISON OF LCA, ALA, AND SMA IN SERA OF 26 MS PATIENTS Type of antibody Serum
LCA
1718 1747 1684 1707 1685 1692 1695 1673 1717 1749 1691 1779 1694
+ +
Total positive
40
40
+ + + 5 LCA+
SMA” (Rat tissue)
ALA”
80 40
40 40 110
9
8
7 =CI>40
LCA-
Negative correlation between LCA and ALA LCA and SMA (rat tissue) LCA and SMA (HEp, cells) Positive correlation between ALA and SMA (rat tissue) SMA (rat tissue) and SMA (HEp, cells) ALA and SMA (HEp, cells) ” These are in IgM class. Note: Antibody titers are expressed as reciprocals.
SMA” (HEp, cells)
=CI<40 P = 0.5875 P = 1.000 P = 1.000 P = 0.0022
P = 0.0019 P = 0.0019
(Cl (‘2)
(MS)
(MS) (MS)
____-
40 80 160 20 40
SMA titer
OF SMA
10
IO
IO
10 40 40
10
IO 20 20
MS brain
10
10
10 40 40
Normal brain
CLASS OF MS
SMA after with
oh THE IgM
of IgM absorption
Uterus
-____
Titer
&ND ALA
40 - ___-.-.
20
40 80 160
RBC pool
TABLE ASD CONTROL
40 40 NT’ 40 NT
ALA titer
5 SER.\
_.._
(91 i
(100) (93)
(‘,;; ) i,
_
Titer
~EFORF
XFTFR
ABSORPII~K
-
10
10 (84)
10
~-.Normal brain
VARIOC;~
preparationa.
- .___ -_ ----.--lymphocyte
_---.
I0
IO (97)
20 190) IO (9.5)
10
MS brain
- --
MIT-H
10
Uterus
of IgM ALA” after absorption with -__.--~. ~-
\h;~
___II_-. “1’0~c. Antibody titers are expressed as reciprocal\. ’ The titers of the ‘41~4 in the IgM cl;\\\ were average< uf rhtlsz detected on IWU MS and two control ” Average percentage of sells giving po\itivc \taining (i.e.. or - I OII the four cell prepurati0n.r. ’ NT. not tested.
1747 1718 1717 2007 2004
Serum
THY TIIERS
.--
-
40 (89,
40 (100) 40 (961
RBC pool
. -.-----
- --
ABSORIL+NIS
: 3 E; 0 L
2 ;
T
5
5
5
w w
AUTOANTIBODIES
IN MULTIPLE
383
SCLEROSIS
absorption with human rbc had no effect (Table 5). The ability of the myometrium, normal and MS brain appeared to be somewhat similar in absorbing out IgM activity. RF was detected in 1126 MS sera, and absorption with latex-coated y-globulin removed the low-titer RF from the serum but only slightly reduced the titers of both SMA and ALA in IgM class from the sera of the eight patients with MS (Table 6). Similar absorption of 2 sera from patients with RA with high-titer RF, reduced the titer eightfold. The ALA activity detected in either IgG or IgM class of the whole serum by indirect immunofluorescence was also found in the corresponding immunoglobulin class fractions, when tests were carried out on sera from three patients with MS and from a healthy person (Table 7). DISCUSSION
We have not found so high an incidence of LCA in sera of MS patients as previous workers (2, 16), but the lack of correlation we have shown between LCA and HLA cytotoxic antibodies is similar to that shown by Kuwert and Bertrams (1). We have found a significantly higher level of ALA in IgM class in MS compared with control sera, and the staining pattern in the immunofluorescence test appears to be similar to the description of Husby and Messner (4) and Searles, Husby, and Messner (5) of the staining of lymphocytes by SLE sera. Antilymphocyte antibodies (ALA) have not been correlated with LCA in the sera of SLE patients (17), of relatives of SLE patients (5), and of patients with IBD (6). Similarly our results did not show any correlation between LCA and ALA. However, we have shown a correlation between ALA and SMA in IgM, and this may point to the occurrence of antigen or antigens of similar specificity in the
THE EFFECT
TABLE 6 OF MS AND RA SERA WITH LATEX ON VARIOUS ANTIBODY TITERS
OF ABSORPTION GLOBULIN
Titer of antibodies” In serum absorbed with latex y-globulin
In unabsorbed serum Sera 1718 (MS) 1695 (MS) 1747 (MS) 1673 (MS) 1694 (MS) 1779 (MS) 1692 (MS) 2230 (MS) 9158 (RA) 9064 (RA)
RF
SMA
ALA (‘%)b
RF
20 20 20 20
40 (78) 20 (63) 20 (91) 10 (52) 20 (83) 20 (58) 40 (96) 40 (94)
SMA 20 10 20 10
ALA (%)” 40 (75) 20 (68) 10 (81) 10 (61) 10 (87) 10 (43) 20 (98) 40 (91) Cl0
Note. Antibody titers are expressed as reciprocals. ” The SMA, and ALA were all in IgM class. I’ Mean percentage of cells staining using one MS and one normal lymphocyte preparation.
384
MCMILLAN,
HAIRE,
TABLE THE
PRESENCE
OF IgG
AND
IgM
ALA
IN WHOLE
AND
MIDDLETON
7 SERUM
AND
IgG
AND
IgM SERUM Faxrroxs
Presence of ALA staining” -.-~ In whole serum at dilution 1120 Serum 1747 (MS) 1692 (MS) 1718 (MS) 2007 CC) 1974 (0 I973 (f.3
G
M
In IgG fraction
In
G
G
M
IgM
fracrion M
-~ +
t
^
+ -
+
^
_.
‘I The results were obtained in duplicate using a normal lymphocyte preparation as antigen.
three antigens used, i.e., lymphocytes, rat composite tissue, and cultured HEp, cells, and absorption studies using human uterus, normal, and MS brain suggest a smooth-muscle antigen specificity. In two of these MS sera, the specificity of the SMA in IgM has been shown to be against a component of cell intermediate filaments (14). Since absorption of sera with pooled-adult and cord rbc did not affect the titers of SMA it is indicated that antibody was not directed against the antigens of the Ii system. The finding that MS lymphocytes were a more sensitive antigen than control cells to detect ALA may indicate an increase of smooth-muscle-type antigen made available in lymphocytes by viral or other stimulation, somewhat similar to the enhancing effect of phytohemagglutinin (PHA) on smooth-muscle antigen in peripheral blood lymphocytes (9). Since RF in serum has been shown to be responsible for false positive IgM staining of viral antibodies in the immunofluorescent technique (18), and IgM staining of measles virus in the sera of MS patients has been described as being caused by a rheumatoid-like factor (19, 20), experiments were carried out to ensure that RF was not responsible for the staining patterns found when testing for ALA and SMA in IgM class. False positive viral IgM staining is only found in the presence of the corresponding viral antibody in IgG class (18, 21). ALA in IgG class was infrequent and indeed ALA in IgM class was shown in the absence of ALA in IgG class. In addition, our results show that ALA was not removed by absorption with latex-coated y-globulin, and ALA in IgM class was only detected in IgM fractions of sera. The slightly higher incidence of ALA and SMA in IgM class in MS patients compared with controls may result from alteration in T-cell activity. A persistent viral infection may cause a loss of suppressor T-cell function or may cause a loss of tolerance to autoantigens of helper T cells. The reaction of ALA with membranes of lymphocytes, with increased activity with patients’ cells, may indicate an alteration of receptors on lymphocytes for IgM in MS. Since the patients in this study were all probable cases in an inactive phase, a further study is warranted of autoantibodies to smooth muscle and subpopulations of lymphocyte membranes in MS patients with active disease.
AUTOANTIBODIES
IN MULTIPLE
SCLEROSIS
385
ACKNOWLEDGMENTS This work has been made possible by the collaboration of many colleagues. The patients were under the care of Dr. J. H. D. Millar, who also read the manuscript. Colonel T. D. Field made available the normal control specimens. Dr. J. D. Merrett, Department of Medical Statistics, The Queen’s University of Belfast, carried out the statistical analyses. Dr. P. V. Shirodaria gave valuable criticism and advice. Mrs. E. E. Russell, Mrs. J. P. Douglas, Mrs. J. Totton, Mrs. C. Sleator, and Mrs. S. A. Kirk provided technical assistance. Miss J. Smyth typed the manuscript. We wish to thank these colleagues. Part of the work carried out in the Department of Microbiology and Immunobiology, The Queen’s University of Belfast, is supported by a Program Grant from the Medical Research Council.
REFERENCES 1. Kuwert, E., and Bertrams, J.. Eur. Neural. 7, 65, 1972. 2. Schocket, A. L., Weiner, H. L., Walker, J., McIntosh, K., and Kohler. P. F., C/in. fmmunol. Immunopatld.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
7, 15, 1977.
Goldberg, L. S., Bresnihan, B., and Hughes, G. R. V., C/in. Exp. fmmunol. 31, 443, 1978. Husby, G., and Messner, R. P.. J. Lab. Clin. Med. 89, 240, 1977. Searles, R. P.. Husby. G., and Messner, R. P., Acta Pathol. Microbial. Scund. 74C, 463, 1977. Henderson, C. A., Greenlee, L., Williams, R. C.. Jr., and Strickland, R. G., Scund. J. Immunol. 5, 837, 1976. Haire, M., &it. Med. Bull. 33, 40, 1977. McMillan, S. A., and Haire, M., unpublished data. Fagraeus, A., Lidman, K., and Biberfeld, G., Nature (London) 252, 246, 1974. Millar, J. H. D., Fraser, K. B.. Haire, M., Connolly, J. H., Shirodaria, P. V., and Hadden, D. S. M., h-it. Med. J. 2, 378, 1971. Mottironi, V. D., and Terasaki, P. I., In “Histocompatibility Testing” (P. I. Terasaki, Ed.), pp. 301-308, Munksgaard, Copenhagen, 1970. Nelson, S. D., and Middleton, D., Ulster Med. J. 44, 48, 1975. McMillan, S. A., and Haire, M.. C/in. Exp. Immunol. 21, 339, 1975. McMillan, S. A., and Haire, M., C/in. Immunol. Immunopathol. 14, 256, 1979. Hannon. R., Haire. M., Wisdom. G. B.. and Neil], D. W., J. Immunol. Methods 8, 29, 1975. Schocket. A. L., and Weiner, H. L., Lancer 1, 571, 1978. Messner, R. P., Kennedy, M. S., and Jelinek, J. G., Arthritis Rheum. 18, 201, 1975. Shirodaria. P. V.. Fraser. K. B., and Stanford, F., Ann. Rherrm. Dis. 32. 53, 1973. Fraser, K. B., Lancrt 1, 91, 1978. Fraser, K. B., Armstrong, M.. Shirodaria. P. V., and Gharpure, M., C/in. Exp. Immunol. 36, 304, 1979. Shirodaria, P. V., Fraser, K. B., Armstrong. M.. and Roberts, S. D., Infer. Immunity 25, 408, 1979.