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Autologous lymphocyte proliferation in multiple sclerosis and the effect of intravenous ACTH.

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Autologous Lymphocyte
ProMeration in Multiple Sclerosis
and the Effect of Intravenous ACTH
Gary Birnbaum, MD, and Linda Kotilinek, BS
~
~
The proliferative response of peripheral blood T cells to autologous non-T cells, a reaction called the autologous
mixed lymphocyte reaction (MLR), was significantly increased in 17 patients with active multiple sclerosis (MS)
compared to age- and sex-matched individuals with other neurological diseases (OND). Following a 10-day course
of intravenous adrenocorticotropic hormone (ACTH) therapy the values were reduced to control levels. No
differences were noted between MS patients and controls in their response to alloantigens. The increased autologous MLR in patients with active MS appeared to result from an increased stimulatory capacity of non-T cells
rather than from an intrinsically greater T cell proliferative potential. ACTH appeared to induce a change in the
populations of circulating non-T cells such that these cells had a decreased stimulatory capacity in both autologous
and allogeneic MLR. The decrease in stimulatory capacity in autologous MLR was, however, significantly greater
than the decrease in allogeneic stimulatory capacity, suggesting a functional decrease of specific non-T cellenriched subpopulations. No significant changes in the numbers of myeloperoxidase-positive(MP+) cells were
noted in the blood of MS patients before and after ACTH therapy. Since the autologous MLR results in generation
of cells that regulate immune responsiveness, the changes noted provide additional evidence for abnormal immune
regulation in MS.
Birnbaum G, Kotilinek L: Autologous lymphocyte proliferation in multiple sclerosis and the effect of
intravenous ACTH. Ann Neurol 9.430-446, 1081
The cause of multiple sclerosis (MS) is unknown, and
treatment has been empirical. Recent work has suggested that viral infection [ S , 271, autoimmune
phenomena [ 3 3 , 371, or a combination of these two
processes [ l , 121 may be important in causing the illness. As a result, immunosuppressive drugs such as
corticosteroids and adrenocorticotropic hormone
(ACTH) have been used to ameliorate symptoms and
shorten the duration of acute attacks [ 2 9 ] . Their
mechanisms of action are, however, not known.
If MS is the result of an autoimmune process, that
is, one in which there is an alteration of selftolerance, it may be possible to demonstrate such
changes using the recently described human autologous mixed lymphocyte reaction (MLR) [19, 281.
This is an in vitro immunological reaction to selfantigens in which T cells proliferate in response to
autologous non-T cells. This reaction demonstrates
specificity and immunological memory and has been
shown to generate populations of regulatory lymphocytes, both suppressors and helpers [ 1 3 , 31, 35, 381,
as well as soluble factors that aid in the initiation of a
variety of immune responses [ 7 , 401.
The autologous MLR is altered in several diseases
with abnormal immune regulation. These are systemic
lupus erythematosus (SLE) [ 2 0 , 321, chronic lymphocytic leukemia [ 3 4 ] , and infectious mononucleosis [24]. In all these diseases the autologous MLR is
decreased. This reduction has been attributed to both
a diminished stimulatory capacity of patients’ B cells
[ 2 0 , 2 4 , 3 2 , 341 and decreased proliferative capacity
of their T cells [ 3 2 ] .
The purpose of our experiments was to quantitate
t h e autologous MLR in patients with active MS and
to determine the effects of ACTH on this immune
reaction.
From the Department of Neurology, University of Minnesota
School of Medicine, Minneapolis, MN 55455.
Received June 20, 1980, and in revised form Oct 1 . Accepted for
publication Oct 4 , 1980.
Materials and Methods
All study patients were hospitalized. Patients with probable
o r definite MS conforming to well-established diagnostic
criteria were admitted to the hospital either in acute (less
than 2 weeks) exacerbation of their illness o r with a clearly
demonstrable progressive increase in neurologcal signs
and symptoms. Hospitalization was for the purpose of
treatment with intravenous ACTH. Blood was obtained on
admission to the hospital prior to initiation of treatment,
Address reprint requests to Dr Birnbaum.
0364-5 134/81/050439-0S$01.25 @ 1980 by the American Neurological Association
439
and again within 2 to 3 hours after completion of a course
of ACTH. Seventeen MS patients were studied, 13 women
and 4 men, who ranged in age from 21 to 51 years. Most
patients were in their late twenties o r early thirties.
Blood was obtained from 22 hospitalized age- and sexmatched patients with other neurological diseases (OND).
This group consisted of 4 individuals with seizures, 4 with
headaches, 2 with lumbar radiculopathy, 2 with syncope,
and 1 each with Huntington’s chorea, Guillain-Bark syndrome, head trauma, idiopathic optic atrophy, femoral
neuralgia, neurofibromatosis, Fabry’s disease and stroke,
idiopathic myelopathy, chronic peripheral neuropathy, and
mental retardation with seizures.
No patients were studied if they had received any drugs
within the past month known to affect immune function,
nor were patients studied who had diseases known to be
associated with immune abnormalities, such as SLE, neoplasia, or acute infections.
Normal individuals could not be tested concurrently
with MS patients and OND controls because of limitations
imposed by the size of each experimental protocol.
ACTH (Acthar, lyophilized; Armour Pharmaceuticals,
Phoenix, AZ 85007), 4 0 USP units diluted in 500 ml of 5%
dextrose solution, was intravenously administered daily
over a 6- to 8-hour period. The drug was given for 10 days
to 15 patients and for 7 days to 2 patients.
Peripheral venous blood was obtained in heparinized
syringes and diluted with an equal volume of phosphatebuffered saline (PBS), p H 7.2. Equal volumes of blood
were obtained before and after ACTH treatment. The diluted blood was layered onto a discontinuous gradient of
Ficoll-Hypaque, specific gravity 1.076 to 1.080, and centrifuged for 40 minutes at 200 g. T h e peripheral blood
mononuclear cells (PBM) accumulating at the gradient
interface were collected. Viability, as assayed by trypan
blue dye exclusion, was greater than 95%. All PBM obtained from MS patients and control groups were frozen
and stored at -80°C or in liquid nitrogen prior to use, so
that pre-ACTH and post-ACTH samples as well as control
samples could be assayed concurrently.
Either unfractionated PBM or T-enriched and Benriched cell populations were suspended in cold (4°C)
medium consisting of RPMI 1640 with 20% fetal calf
serum (FCS) (Grand Island Biological Company, Grand
Island, NY) and preservative-free I % heparin (Weddel
Pharmaceuticals, Ltd., London, England). Cells were at a
concentration of 20 to 24 x lo6 per milliliter. An equal
volume of cold RPMI 1640 medium containing, in addition
to these ingredients, 20% dimethyl sulfoxide (Sigma
Chemical Company, St. Louis, MO) was added a drop at a
time to the cell suspension. Cells were then quickly cooled
at a rate of 1°C per minute, using a Linde cell freezing unit,
or were placed in a deep-freezer at -80°C. Stored cells
were kept either at -80°C or in liquid nitrogen.
Cells were thawed by immersion in water at 37°C and
then were quickly diluted by adding, drop by drop, a fivefold greater volume of RPMI 1640 and 20% FCS. Cells
were washed several times in medium and either used in
culture at that point or fractionated into T and B cellenriched populations.
The technique of Brown et a1 [6] was used for prepara-
440 Annals of Neurology Vol 9 No 5 May 1981
tion of enriched populations of T cells and B cells. Briefly
described, fresh sheep red blood cells (SRBC), less than 2
weeks old, were treated with Vibrio cbolerae neuraminidase
(Behring Diagnostics, Somerville, NJ 98876), 0.4 U/ml, in
PBS for 1 hour at 37°C. Equal volumes of washed SRBC,
at a concentration of 1 x 10’ cells/ml, were added to
PBM, 10 x lo6 cells/ml, suspended in RPMI 1640 with
20% SRBC-absorbed FCS. Cells were incubated for 10 minutes at 37°C. The cell mixture was then centrifuged on a
Ficoll-Hypaque gradient at ambient temperature at 450 g
for 30 minutes. Cells collecting at the interface were
non-T cells. T h e pellet was T cell-enriched. SRBC in the
T-enriched pellet were lysed by brief exposure ( 5 seconds)
to distilled water. T h e purity of the B cell population was
determined by reacting cells with fluoresceinated, polyvalent rabbit antihuman Ig (Miles Laboratories, Elkhart, I N
465 14) for 30 minutes at 4°C and counting 150 to 200 cells
under ultraviolet light. T cell purity was determined by
taking 50 pI of the T cell-enriched pellet prior to SRBC
lysing, centrifuging the cells, and incubating them at 4°C
for 3 to 4 hours. Twenty microliters of fluorescein diacetate
(1 mg/ml in acetone) was added to the gently resuspended
T-enriched cells, and the percentage of rosetted cells was
determined after counting at least 150 to 200 cells under
ultraviolet light. T h e purities of both populations were almost always greater than 85 to 90%. Since rabbit antiserum
to human Ig will react with cells having either or both surface Ig and Fc receptors, we considered our non-T cells to
be a mixture of B cells, monocytes, and null cells.
Since our non-T cell preparation consisted of mixtures
of different cell types, we utilized a myeloperoxidase
staining technique to quantitate the number of monocytes
present in our non-T cell fractions. T h e technique of
Kaplow was used [16]; briefly described, cells dried onto
slides were fixed in 10% formal-ethanol and then rinsed in
tap water. Cell nuclei were counterstained for 2 minutes in
freshly prepared Giemsa solution. The percentages of
myeloperoxidase-positive (MP+) cells were determined by
examining at least 200 cells under oil immersion for the
presence of dark-brown granules.
Tissue culture medium consisted of RPMI 1640 with
10% pooled, heat-inactivated human AB serum, penicillin,
streptomycin, and L-glutamine. T-enriched responder cells
were suspended at a concentration of 1 x lo6 cells/ml.
Varying effector/stimulator ratios were used, ranging
from 1 : 1 to 1:4; this was done to obtain dose/response
curves of the sensitivity of different effector cell populations to the stimulatory capacities of autologous and allogeneic cells. Ratios were chosen that gave proliferative
responses below maximal plateau values, increasing the
sensitivity in our assays. In general, effector/stimulator
ratios were 1: 2 in allogeneic MLR and 1 :4 in autologous
MLR. Allogeneic stimulation was provided by a pool of
PBMs from 5 unrelated donors. All stimulator cells were
x-irradiated with 3,000 rads using a cesium 127 source.
One-tenth milliliter each of effector cells and stimulator
cells were added to round-bottomed, multi-well tissue
culture plates (Linbro Division, Flow Laboratories, Inc.,
Hamden, C T 06517). Wells were set up in triplicate and
cultured for varying periods at 37°C in a humidified atmosphere of 5% carbon dioxide and 95% air.
I
15 c
- Pre-ACTH
--- Post -ACTH
--- MS
OND
I
I
t
't
2L.
_L
6
8
1
10
I
13
6
1
I
8
10
I
13
Days
Days
F i g 1. Comparison of autologous MLC proliferative responses
in M S patients before and after A C T H therapy. The abscissa
represents days in culture; the ordinate is the log,, of counts per
minute of :%H-thymidine.Arrows indicate the standard error.
Eighteen hours before the end of culturing, 2 pCi of
"-thymidine, specific activity 6.7 Ci/mmol (New England
Nuclear, Boston, MA) was added to all wells. Cells were
harvested onto glass-fiber filter paper with a multisample
cell harvester. The amount of isotope incorporated was
determined by counting samples in a liquid scintillation
counter.
Counts per minute of tritiated thymidine incorporated
by unstimulated cells were subtracted from the counts per
minute incorporated by stimulated cultures. These corrected counts were then converted to logarithms of base
10. All computations were performed on the transformed
variables. Means and standard errors were calculated. Means
were compared by the paired Student t test and by t test for
independent samples.
T h e data presented iq Figures 1 through 4 represent
mean values obtained from the groups of OND and MS
patients; those in the Table represent individual MS and
OND patients.
Results
Effect of ACTH on Numbers of PBMs
Equal volumes of peripheral venous blood were obtained from MS patients immediately before initiation of ACTH therapy and within 3 hours after completion of the last dose. PBMs were obtained from
these samples using the same standard technique.
The numbers of PBMs obtained in pre-ACTH and
post-ACTH samples were compared. A significant
decrease in PBMs collecting at gradient interfaces
was noted in post-ACTH blood samples. Pre-ACTH
samples had a mean value of 1.25 x lo6 PBMs/ml,
F i g 2. Comparison of autologous MLC proliferative responsej
in MS patients (beforeA C T H therapy) and patients with
OND. Abscissa and ordinate values are same as i n Figure 1.
5
4
z
0
CSI
0
J
3
P r e - ACTH
--- Post ACTH
-
2
I
1
I
I
6
8
10
13
Days
F i g 3. Comparison of the allogeneic stimulatory capacity of MS
patient B cells before and after A C T H , using T cells from patients with OND as responders. Abscissa and ordinate values
are same as in Figure 1 .
Birnbaum and Kotilinek: Autologous Lymphocyte Proliferation in MS
441
........... .
5
-
T+B,, Pre ACTH
Past T i P r e Ex
TiB,, Post ACTH
Pre T i P o s t 8
,
Effects of A C T H on Myeloperoxidase-positive Cells
i n Peribheral Blooda
Percentage of
Patients
MS 1
Pre-ACTH
z!u
Post-ACTH
4-
MS 2
Pre-ACTH
m
Post-ACTH
3
MP+ Cells
PBM
T-enriched"
B-enriched
22
35
3 (95)
10 (86)
63 (95)
62 ( 9 7 )
30
67
4 (91)
30 (91)
50
59
32 ( 7 7 )
26 (89)
28
64
18 (91j
7 0 (100)
87 (93)
37
4 (87)
19 ( 8 3 )
50 ( 9 5 )
45 ( 9 5 )
17
20
3 (89)
5 (81)
44 ( 9 5 )
52 ( 9 5 )
29
24
11
32
33
26
15
15
14 ( 8 6 )
3 (87)
2 (74)
8 (90)
3 (73)
8 (83)
4 (93)
1(95)
MS 3
Pre-ACTH'
Post-ACTH
3-
MS 4
Pre-ACTH
Post-ACTH
7 (89)
MS 5
Pre-ACTH
Post-ACTH
Days in Culture
Fig 4. Comparison of the autologous proliferative responses of
pre- and post-ACTH T cells with pre- and post-ACTH B
cells. AbscisJa and ordinate values are same as in Figure 1 ,
MS 6
Pre-ACTH
Post-ACTH
MS 7
Pre-ACTH
Post-ACTH
while post-ACTH samples had a mean value of 0.89
x lo6 PBMs/ml. This difference was significant at the
p < 0.01 level.
Intravenous ACTH thus resulted in a significant
decrease in accumulation of PBMs at Ficoll-Hypaque
gradient interfaces. This finding was most likely the
result of a peripheral blood lymphopenia [20].
Since steroids are not cytotoxic in humans [8, 11,
181, ACTH may have induced a redistribution of circulating PBMs from the peripheral blood.
Proliferative Response t o Autologous B Cell Antigens
One-way autologous mixed lymphocyte cultures
(MLCs) were concurrently established from preACTH and post-ACTH blood samples from the
same MS patient and from an age- and sex-matched
control with OND. Cultures were harvested after 6,
8, 10, and 13 days of incubation. Results are shown in
Figures 1 and 2. The kinetics of thymidine incorporation were the same in all populations, reaching
peak values in allogeneic MLCs after 6 days of culture and in autologous MLCs after 8 days. The magnitude of the autologous responses was, however,
higher in the MS pre-ACTH samples than in the
control groups (see Fig 2), reaching statistical
significance on day 8 (p < 0.04). Following ACTH
therapy the autologous proliferative responses of MS
patients fell, reaching levels almost identical to those
of the control group (see Fig 1). The differences in
responses between pre-ACTH and post-ACTH samples were statistically significant for days 6 (p <
442 Annals of Neurology Vol 9 No 5 May 1981
OND
OND
OND
OND
OND
OND
1
2
3
4
5
6
17
46 (97)
50 ( 9 7 )
64 ( 9 7 )
74 (100)
80 (100)
25 (96)
53 ( 9 7 )
43 ( 9 3 )
"Peripheral blood was obtained from MS patients before and after
ACTH therapy and from age- and sex-matched patients with
OND. PBMs, unfractionated or separated into T-enriched or Benriched cell populations, were assayed for the number of MP+
cells.
"Numbers in parentheses represent the 9% purity of the isolated
cell populations: 96 of cells having SRBC rosettes for the Tenriched cells and % of cells having cell surface Ig for the Benriched cells.
"This patient was taking birth control pills at the time of admission
to the hospital.
0.001), 8 (p < 0.003), and I 0 (p < 0.05). Autologous
proliferation thus appeared to be increased in patients with progressive or active MS, and this response returned to control values after intravenous
ACTH therapy.
Proliferative Response t o Alloantigens
The proliferative responses to a constant pool of allogeneic PBMs were compared using T-enriched
cells obtained from MS patients before and after
ACTH therapy and T-enriched cells obtained from
patients with OND. Responses were measured in
one-way allogeneic MLCs. Cultures were harvested
6, 8, 10, and 13 days after their initiation. Neither
the degree of proliferation nor the kinetics of the response differed in any of the three groups; these
findings suggested that the increased autologous
MLR seen in pre-ACTH blood samples did not result
from any generalized increase in T cell reactivity, and
that the decrease in autologous MLR observed after
ACTH therapy was not the result of a nonspecific
decrease in T cell responsiveness.
Allogeneic Stirnulatory Capacity of Pre-ACTH
and Post-A C T H B-en ricbed cells
T o investigate the possibility that the B-enriched cell
fraction may be the population affected by ACTH,
we measured the capacity of B-enriched cells obtained from pre-ACTH and post-ACTH blood samples to evoke a proliferative response in allogeneic
T-enriched cells from patients with OND.
Effectorlstimulator ratios of 1 : 1 and 1 : 2 were
used, and cultures were harvested 6, 8, 10, and 13
days after the start of culture. Results of the proliferative responses obtained with effectoristimulator
ratios of 1 : 1 are shown in Figure 3 . Results obtained
with ratios of 1 : 2 were analogous. Post-ACTH Benriched cells demonstrated a statistically significant
decrease in their ability to induce a proliferative response cornpared to pre-ACTH B-enriched cells
from the same individuals. These differences were
most marked on day 6 (p < 0.009) and day 8 (p <
0.0 5 ) .
Previous work [15, 181 demonstrated that the autologous MLR is much more sensitive to the suppressive effects of corticosteroids than is the allogeneic
MLR. To determine whether relatively selective suppression of autologous reactivity was induced by
ACTH in patients with MS, we compared the means
of the differences noted in pre-ACTH and postACTH autologous MLC with the means of differences noted in allogeneic MLC in which B-enriched
cells from pre-ACTH and post-ACTH blood samples
were stimulators. Using paired t test analyses of cultures established with effector/stimulator ratios of
1 : 1, the decrease in autologous reactivity on days 6
and 8 was significantly greater than was the decrease
in allogeneic reactivity measured on those same days
(p < 0.02 for both days). Comparing decreases in
autologous MLCs with decreases in allogeneic MLCs
established at effector/stimulator ratios of 1:2 gave p
values for the differences on days 6 and 8 of culture
of <0.01 and <0.005, respectively. ACTH thus appeared to induce relatively selective suppression of
autologous MLR.
This selective suppression of autologous reactivity
could have been related to the lesser proliferative response induced by autologous cells compared to allogeneic cells. In other words, a weaker proliferative
response may have been easier to suppress than a
stronger one. We thus compared the mean counts
per minute of "H-thymidine incorporated by pre-
ACTH autologous MLC on days 6 and 8 of culture
with the mean counts per minute incorporated by
allogeneic MLC on those same days of culture. Allogeneic MLCs at effectorlstimulator ratios of both
1 : 1 and 1 :2 were used in the analysis. After 6 days
of culture, both groups of allogeneic MLCs proliferated to a significantly greater degree than did autologous MLCs (p < 0.005). By day 8, however,
there were no significant differences in the amount of
:iH-thymidine incorporated by both autologous and
allogeneic MLCs. Since significantly greater suppression of autologous than of allogeneic MLCs was still
noted by day 8 of culture, it is unlikely that this
selective suppression was just the result of a weaker
proliferative response.
The proliferative capability of T-enriched cells
obtained from pre-ACTH and post-ACTH samples
was measured in response to allogeneic B-enriched
cells from OND patients. The protocol was the same
as that described for OND T-effectors. In accord
with the observations made using pooled allogeneic
normal cells, no differences were noted in the allogeneically stimulated proliferative capacities of
T-enriched cells obtained before and after ACTH
therapy.
Autologous Stirnulatory Capacity of Pre-ACTH
and Post-ACTH B-enricbed Cells
While no change in the reactivity to alloantigens of
T-enriched cells after ACTH therapy could be demonstrated, it was nevertheless possible that ACTH
selectively affected the reactivity of these cells to autologous antigens. We therefore cultured pre-ACTH
and post-ACTH T-enriched cells from 5 patients for
6, 8, 10, and 13 days with either pre-ACTH or postACTH autologous B-enriched cells. The T/B ratio
was 1:4. Results are shown in Figure 4 . Pre-ACTH
B-enriched cells were able to induce equivalent proliferative responses in both pre-ACTH and postACTH T-enriched cells, while post-ACTH Benriched cells induced significantly less proliferation
in both pre-ACTH and post-ACTH T-enriched cell
populations (p < 0.005 on day 8 , p < 0.05 on days 6
and 10). ACTH thus did not affect T-enriched cells
but appeared to affect the autologous stimulatory capacity of B-enriched cells. These data also suggest
that the increased autologous MLR seen in patients
with active MS results from an increased non-T cell
stimulatory capacity rather than an intrinsically
greater T cell proliferative capacity.
Effect of A C T H on MonocyteJ
Several reports have described a marked depletion of
circulating monocytes following administration of
steroids [9, 101. This effect was thought to be one
of the important antiinflammatory actions of these
Birnbaum and Kocilinek: Autologous Lymphocyte Proliferarion in M S
443
drugs. We (unpublished observations) and others [3]
have noted that one of the major classes of stimulatory cells in autologous MLR is the MP+ monocyte. We thus quantitated the numbers of MP+ cells
in gradient-purified PBMs of 7 MS patients before
and after a 10-day course of ACTH and compared
them to the number found in 6 patients with OND.
Results are shown in the Table. Prior to ACTH therapy the percentage of MP+ cells in the PBM fraction
in 6 of 7 MS patients was the same as in the control
group. (The 1 patient with elevated MP+ cells before
ACTH therapy was taking birth control pills.) After a
10-day course of ACTH, the percentage of MP+
cells in PBM increased in 4 of 7 patients and was unchanged in the remaining 3 (see the Table). Tenriched and B-enriched fractions were then prepared from these same patients’ PBMs and from the
PBMs of 6 patients with OND. As seen in the Table,
the percentage of MP+ cells increased in the Tenriched fraction in 4 out of 7 patients following
ACTH therapy, while the percentage of MP+ cells in
the B-enriched fraction either increased slightly or
did not change. The purity of pre-ACTH, postACTH, and control group T and B cell-enriched
populations was similar, indicating that the changes
we observed were not due to faulty separation techniques. It should be noted that the populations of
Is+ cells consisted of both monocytes (MP+) and B
cells (MP-), the monocytes being positive because
of having receptors for the heavy chain (Fc) portion
of Ig.
Intravenous ACTH thus either produced little
change or resulted in an increase in MP+ cells in the
PBM fraction of MS patients’ blood. In no instance
was the number of MP+ cells reduced. When present, the increased percentage of MP+ cells was most
prominent in cells separating with the T-enriched
fraction. The decrease in autologous MLR after
ACTH therapy therefore cannot be explained on the
basis of loss of stimulator cells.
Discussion
We have demonstrated that patients with active MS
have an increased proliferative response in autologous MLR compared to an age- and sex-matched control group of patients with OND. Following a course
of intravenous ACTH therapy the autologous MLR
in the MS patients was reduced to the range seen in
the control group. This effect was antigen specific, as
ACTH had no significant effect on the proliferative
response to alloantigens. The cell populations responsible for the increased autologous response appeared to be in the B-enriched non-T cell fraction.
An important stimulatory cell population of the autologous MLR, MP+ monocytes, was the same in
pre-ACTH blood samples of MS patients and of
444 Annals of Neurology
Vol 9
No 5
May 1981
controls. Following ACTH therapy there was either
no change or an increase in MP+ cells in peripheral
blood, particularly in the T-enriched fraction.
T cell proliferative responses in vitro to autologous
non-T cell antigens have been described in both experimental animals 1361 and humans [19, 281. These
responses have many of the characteristics of an
immune reaction, possessing both immunological
memory and specificity [39]. The in vivo role of the
autologous MLR is, however, not known. In vitro,
this reaction generates populations of cells that appear to have a variety of regulatory functions, either
as suppressor cells [3 1, 361 or as helper cells [ 13, 381,
depending on the assays used. More recently,
Chiorazti et a1 [7] and Weksler et a1 [40] have demonstrated that soluble factors generated in the course
of autologous MLR have the capacity to “help” in
differentiation of B cells [7] and in induction of T cell
cytotoxicity responses [401.
Several laboratories have demonstrated a decreased autologous MLR in diseases involving alteration of immune regulation. These diseases include
SLE [20, 321, chronic lymphocytic leukemia [34], and
infectious mononucleosis [24]. The cell type (or
types) responsible for the decreased response in
these diseases is not known but appears in part to be
a non-T cell [20,24,32,34]. MS is, to the best of our
knowledge, the first disease described in which the
autologous MLR is increased.
Several investigators have noted changes in suppressor T cell populations during exacerbations of
MS [2, 141. Such changes could not be demonstrated
in our system; rather, the increased autologous MLR
we noted appeared to result from an increased stimulatory capacity of non-T cells. This increased capacity could have resulted from (1) a qualitative or
quantitative increase in stimulatory cell surface antigens, (2) loss of a population of non-T suppressor
cells, or ( 3 ) an increase in antigen-presenting cells.
While there is no direct evidence for the possibility
that MS B cells possess unique antigens not present
on normal cells, it is possible that chronic viral infection, such as has been postulated to occur in MS [ 1,
121, could result in the appearance of “new” cell antigens. An example of such a phenomenon is the expression of viral-specific antigens on the surfaces of
lymphocytes infected with Epstein-Barr virus [26]. In
addition, certain B cell alloantigens occur with particularly high frequency in patients with MS [41, 421.
Similar alloantigens occur in only a small number of
individuals without MS, suggesting that B lymphocytes in MS patients possibly possess a relatively
unique constellation of surface antigens. We were
not able to demonstrate major changes, either before
or after ACTH therapy, in the total non-T cell
population or in the number of MP+ monocytes pres-
ent in MS patients’ peripheral blood. These observations make it unlikely that our findings resulted
from a nonspecific change in non-T cell populations,
and suggest that the alterations in autologous MLR
may result from changes in antigen-specific cell
populations.
The suppressive effects of monocytes on a variety
of immune responses is non-antigen specific [21,
221. Such cells demonstrate, however, great antigen
specificity in their role as antigen-presenting cells,
that is, as cells that interact with antigen in such a way
as to allow lymphocytes-usually
T cells-to bind
with the material and induce an immune response.
This finding has been especially well shown in strains
of antigen responder-nonresponder animals [4, 301.
In such instances, effective presentation of particular T-dependent antigens to responder T cells is
dependent on the presence of monocytes bearing a
particular constellation of major histocompatibility
complex (Ia) antigens on their cell surfaces [30]. Patients with active MS could have increased numbers
of autologous-antigen specific monocytes in their
blood, allowing for more efficient presentation of
such antigens to T cells and thus inducing greater T
cell proliferation. ACTH could then cause redistribution of antigen-specific helper cells from the peripheral blood, induce changes in cell surface membranes such that antigen presentation is less effective,
or result in the appearance of immature antigenpresenting cells in the peripheral blood. Any of these
situations could result in less efficient helper activity and produce decreases in the autologous MLR.
O u r studies have demonstrated that ACTH had a
relatively specific suppressive effect on the autologous MLR as compared to the allogeneic MLR. Ilfeld
et a1 [ 151 noted specific suppression of the autologous MLR upon addition of hydrocortisone in vitro,
and Katz and Fauci [ 181 noted relatively greater suppression of autologous MLR than of allogeneic MLR
after both in vitro and single-dose in vivo hydrocortisone administration. The latter investigators postulated that the decreased response was due to changes
in the T cell population; in fact, they noted that poststeroid B-enriched cells had a greater stimulatory
effect in both autologous and allogeneic MLC. There
are several possible explanations for the differences
between our results and those of Fauci and his coworkers. O n e is that injection of ACTH results in the
release of a number of different steroids, including
androgens, each possibly having different effects on
lymphocyte subpopulations. More important, the
chronic administration of ACTH, with repeated steroid release, allows for reequilibration of lymphocyte
subpopulations, a phenomenon that would not be
seen in Fauci’s experiments. In support of this
hypothesis has been the recent paper by Kaschka and
Hilgers [ 171. These workers evaluated changes in
lymphocyte subpopulations in MS patients receiving
ACTH over 28 days and noted acute changes in T
cell populations, similar to those of Fauci et al, early
in the course of therapy with a gradual return in lymphocyte populations to pre-ACTH values after about
10 days. These changes were similar to those noted in
myasthenia gravis patients receiving ACTH, suggesting that the alterations are not disease specific.
As noted, the role of the autologous MLR in vivo
and in MS is not known. It has, however, been shown
[13, 381 that a variety of immunoregulatory cells,
both helper and suppressor, are generated in the
course of this reaction. The observation of Levitt et a1
[23] that MS patients demonstrate increased T cell
helper activity for B cell differentiation could be
explained by our observations of increased autologous MLR in such patients. There is thus increasing
evidence in support of the hypothesis that immune
regulation is impaired in MS.
Supported in part by Grant R G 1179-A-3 from the National Multiple Sclerosis Society and by Grant 7R01-NS-14787 from the
National Institutes of Health. Doctor Birnbaum is the recipient of
Research Career Development Award 7K04-NS004 12 from the
National Institutes of Health.
We thank D r Barry Handwerger for his critical reviews of this
paper, D r Ruth Loewenson for her expert statistical analysis, and
Ms Stephanie Daily for her secretarial help.
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