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Cellular immunoregulatory mechanisms in the central nervous system Characterization of noninflammatory and inflammatory cerebrospinal fluid lymphocytes.

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Cellular Immunoregulatory Mechanisms
in the Central Nervous System:
Characterization of Noninflammatory
and Inflammatory Cerebrospinal
Fluid Lymphocytes
Makoto Matsui, MD," Kazuhiro J. Mori, PhD,? and Takahiko Saida, MDS
~~~
Dual-label flow cytometric analysis of cerebrospinal fluid (CSF) and blood lymphocytes with combinations of monoclonal antibodies such as CD4 plus CD45R or Leu8, and CD8 plus CD11b was performed in 37 patients with noninflammatory neurological diseases (NINDs) to clarify the differences in cellular immunoregulatory mechanisms present in
the central nervous system (CNS) and in the systemic circulation. In the CSF of patients with NINDs, the paucity of
CD4+CD45R' and CD8+CD1l b + cells was striking, whereas the same subsets accounted for substantial proportions in
the blood. CD4+CD45R- and CD4+Leu8- cells as well as CD8+CDllb- cells increased in the CSF when compared
with those in the blood. Seven patients with active multiple sclerosis (MS) and 10 patients with other inflammatory
diseases in the CNS (CNS-infl)were also studied. Patients with active MS were characterized by a consistent increase in
percentage of CD4+CD45R- cells in the CSF, whereas an increase of CD4-CD45R+ cells in the CSF was a feature of
the patients with CNS-infl, when compared with patients with NINDs. These findings indicate that the CNS is
routinely surveyed by particular subsets of lymphocytes different from those in the blood, and cellular immune
reaction in the CNS varies according to the types of CNS inflammatory conditions.
Matsui M, Mori KJ, Saida T. Cellular immunoregulatory mechanisms in the central
nervous system: characterization of noninflammatory and inflammatory cerebrospinal
fluid lymphocytes. Ann Neurol 1990; 27647-651
The central nervous system (CNS) has long been
thought to be an immunologically privileged site because of its relative unresponsiveness to allografts and
inoculated pathogens C 11. The blood-brain barrier
(BBB) isolates the CNS from the systemic circulation,
so that only a few lymphocytes and very low levels of
immunoglobulins are known to exist in the cerebrospinal fluid (CSF) in a noninflammatory state. Accumulating evidence has, however, indicated that lymphocytes
in the CSF have unique immunological characteristics
in comparison with those in the peripheral blood. For
example, CSF lymphocytes proliferate poorly in response to plant lectins in inflammatory CNS diseases,
whereas the blood lymphocytes examined simultaneously proliferate well C2, 31. It has been proposed
that the CNS is routinely patrolled by activated T lymphocytes [4}. These findings indicate that a particular
population of T lymphocytes that are comparunentalized within the CNS play an important role in CNS
immunoregulation. In the present study, we attempted
to characterize T cell subsets in CSF from patients with
both inflammatory and noninflammatory neurological
disorders and compare the subsets with those in peripheral blood by using monoclonal antibodies
(MoAbs) against CD45R C51 and Leu8 16, 71 antigens
for CD4+ lymphocytes, and one MoAb against
C D l l b 18, 91 antigen for CD8+ lymphocytes.
From the *Department of Neurology, Faculty of Medicine, Kyoto
University, Kyoto; the ?'Department of Biology, Faculty of Science,
Niigata University, Niigata; and the %Department of Neurology,
Utano National Hospital, Kyoto, Japan.
Received Jun 30, 1989, and in revised form Nov 14. Accepted for
publication Dec 21, 1989.
Address correspondence D~Matsui, do D~H, L,weiner, Center
for Neurologic Diseases, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.
Material and Methods
Patients
Thirty-seven patients with noninflammatory neurological diseases (NINDs) were studied to establish reference levels for
the percentage of T-cell subsets in the CSF, as well as in the
blood. This group consisted of 24 men and 13 women be-
Copyright 0 1990 by the American Neurological Association
647
tween 22 and 72 years of age (mean 45.4 yr), including 9
patients with spinocerebellar degeneration and other related
disorders, 7 with motor neuron disease, 6 with noninflammatory neuropathy, 4 with remote cerebrovascular accidents,
4 with myopathy, 2 with myeloradiculopathy due to cervical
spondylosis, and 5 with other illnesses. N o one in this group
received corticosteroids or immunosuppressive agents. The
number of CSF cells was less than 3/mm3 in every patient in
the NINDs group. To clarify alterations in the CSF with
inflammation, two groups of patients were examined. Seven
patients with relapsing-remitting type of multiple sclerosis
(MS group) were studied during the active stage of the disease, which was within 2 weeks of new neurological symptoms or worsening of previous ones, or within 2 weeks of
peak deficit. They were patients with clinically definite or
laboratory-supported definite MS, according to Poser's criteria [lo), ranging in age from 32 to 49 years (mean 39.9).
Four showed a CSF pleocytosis (>5/mm3). Three of these 4
and 1 other patient in this group were being treated with
corticosteroids. The other group consisted of 10 patients
with inflammatory CNS diseases other than MS (CNS-infl),
such as aseptic meningitis (3 patients), serous meningeal
reaction to nonsuppurative pachymeningitis (2), meningoencephalitis (2), transverse myelitis (l),lupus meningitis (l),
and syphilitic optic papillitis (1). The last 3 patients were
receiving corticosteroids at the time of study. These patients
were between 17 and 64 years of age (mean 34.0), and were
associated with a slight to marked increase in number of CSF
mononuclear cells (MNCs; 3-2000/mm3).
Cell Preparation, Staining and Flow Cytometry
MNCs were prepared on the same day from the CSF obtained by atraumatic lumbar puncture and from the blood, as
described previously [ll]. In brief, MNCs separated from
the heparinized venous blood by Ficoll-Paque density-gradient centrifugation, or those collected from 8 to 16 ml of the
CSF by a low-speed spinning at 4"C, were suspended in
RPMI-1640 media supplemented with 10% fetal calf serum.
To classify T-cell subsets, MoAbs such as fluorescein-conjugated CD3 (OKT3), CD4 (OKT4 or Leu3a), CD8 (OKT8 or
Leu2a), CD19 (Leul2), anti-HLA-DR, CD45R (Leul8)
[12], phycoerythrin (PE)-conjugated CD4 (Leu3), CD45R
(2H4) [5], Leu8 [6], C D l l b (Leul5) [8}, and in a few instances, CDw29 (4B4) 1131 were studied. The OK series
antibodies were purchased from Ortho Pharmaceuticals
(Raritan, NJ); the Leu series and anti-HLA-DR antibodies
were from Becton Dickinson (Mountain View, CA); 2H4
and 4B4 antibodies were from Coulter Immunology
(Hialeah, FL). The MNCs were single-stained with CD3,
CD8 (OKT8), CD19, or anti-HLA-DR MoAb, or doublestained with combinations of the previously mentioned antibodies such as CD4 plus CD45R or Leu8, and CD8 plus
C D l l b , and less frequently CD4 plus CDw29. All stains
were carried out at 4°C for 30 minutes. Flow cytometry of
MNCs was performed by means of an Ortho Spectrum 111
(Ortho Diagnostic System, Westwood, MA). Monocytes and
macrophages were gated out on the cytogram of the MNCs.
At least 250 celldMoAb from the CSF and more than 5,000
cells/MoAb from the blood passed through a Spectrum 111.
In a preliminary study on blood samples, the results were
648 Annals of Neurology Vol 27 N o 6 June 1990
Percentages of T-cell Subsets in Cerebrospinal Fluid and
Peripheral Blood in Patients with NoninfEammatory
Nelrrological Diseases"
CSF
CD3+
CD4+CD45RCD4+CD45R+
CD4-CD45R'
CD4 'Leu8CD4+Leu8+
CD4-Leu8'
CD8+
CD8+CDllbCD8+CD1l b f
CD8-CDllbt
PB
* 9.9;
88
55 f
1&
5 5
26 f
24 2
10 f
28 +24 t
1 +4 ?
69.4 f 7.5
26.7
7.0
17.1 & 5.0
43.3
7.1
8.6 f 3.3
33.1 +- 7.3
8.2
30.3
24.7 5 5.9
17.7 .+. 5.5
8.7 2 3.6
20.0 ? 5.2
*
*
8.9
1.4'
5.5'
9.0b
9.8'
5.8'
7.0d
5.7d
*
l.lC
4.6'
"All values are expressed as mean
r
rt
0.381
0.236
0.261
0.181
0.061
-0.049
0.168
0.341
0.301
0.120
0.453
standard deviation. Exarnina-
tion by dual-labeling with CD8 and CDllb monoclonal antibodies
were conducted in 10 of the 37 patients.
bSignificantly increased (p < 0.001), as compared with PB.
'Significantly decreased (p < 0.001), as compared with PB.
dSignificantly increased (p < 0.05), as compared with PB.
Statistical analysis of the data was performed by the Student's t test.
Correlation coefficient ( r ) was evaluated by the least-square analysis.
CSF
=
cerebrospinal fluid; PB
=
peripheral blood.
identical between OKT4 plus 2H4 and Leu3a plus Leu18
stains (data not shown). All results were expressed as percentage of each subset to total lymphocytes. Statistical analysis of the data was performed with the Student's t test. Correlation coefficient (r) of each lymphocyte subset between the
CSF and the blood was computed by least-square analysis.
Relationships between the T-cell subsets in the CSF or in the
blood were also analyzed using the same method.
Results
The proportions of T-cell subsets in a noninflammatory state are shown in the Table. The level for each
subset in the CSF was significantly different from that
in the blood. The percentage of CD19+ B cells was
significantly lower in the CSF than in the blood (3%
versus 10% as the mean percentage, p < O.OOl),
whereas the proportions of HLA-DR antigen-positive
(Ia+) lymphocytes were not different between the CSF
and the blood (15% versus 16.9%). Lymphocytes
bearing CD45R antigens were sparse in CSF, as compared with those in blood (6% versus 60.4%). Almost
all the cells in the CD4+ population in the CSF lacked
CD45R antigen, whereas half of the same population
possessed Leu8 antigen. The CD 11b antigen-bearing
cells in the CSF were significantly fewer than in the
blood (5% versus 28.7%), so that most of the CD8+
cells in CSF were CD1 Ib-. A preliminary study indicated that the majority of the CD4+ lymphocytes in
the CSF were CDw29+ (data not shown). No particular subset showed a significant correlation in percentages between CSF and blood (see Table). With respect
to the relationships between the subsets within the
CD4 + population, a significant correlation was observed between the CD4+CD45R- and the CD4+
Leu8- cells in the CSF (Y = 0.699, p < 0.01). In the
blood, the CD4+CD45R- population correlated with
the CD4+Leu8- (r = 0.686, p < O.Ol), while the
reciprocal CD4 +CD45Rf population correlated with
the CD4+Leu8+ (r = 0.670, p < 0.01), and inversely
with the CD4+Leu8- (Y = -0.671,p < 0.01). These
results indicate that a substantial proportion of the
CD4' cells in the blood expressed CD45R and Leu8
antigens simultaneously, whereas such cells were rare
in the CSF. In contrast, the CD4+CD45RP population
overlapped with the CD4+Leu8- in CSF as well as in
blood. Since approximately 80% of the CD4+ cells in
the blood were positive for Leu8, the CD4+CD4JRp
Leu8 - population appeared to be larger in CSF than in
blood.
Examination of CSFs obtained from the patients
with active MS revealed that the percentage of
CD4 +CD45R- lymphocytes significantly increased
(73 ? 4, p < O.OOl), while the patients with CNS-infl
showed a significant increase in percentage of the CSF
CD4-CD45R+ cells (18.9 & 8.9, p < 0.001) when
compared with the patients with NINDs. Neither CSF
pleocytosis nor corticosteroid treatment appeared to
be linked to any particular alterations in CSF subsets
(Figure). The CSF CD4+CD45R+ cells were consistently few; the mean percentages for these cells were
1.5% and 1.6% in the groups of patients with active
MS and CNS-infl, respectively. The proportions of the
other T-cell subsets in CSF were equivalent to those
observed in the patients with NINDs (data not
shown).
Discussion
A prominent feature of the present study was the
paucity of CD45R and C D l l b antigen-bearing cells
in the CSF from neurological patients with noninflammatory states, in contrast to a relative preservation of
the Leu8 antigen. The CD4+ lymphocytes, which simultaneously express high densities of CD45R antigens, have been reported to function as inducers of
suppressor T cells 151, while the C D 8 + C D l l b + cells
have been found to be suppressor cells 181. In this
context, the CNS could be a hazardous site, susceptible to immune-mediated damage due to the lack of
suppressor systems. However, a substantial number of
Leu8 antigen-bearing CD4 + lymphocytes, proposed
to be suppressor-inducer cells [6}, were observed in
the CSF. A recent study showed that there is suppressor activity in CD8+ cells, irrespective of the presence
or the absence of the CD11 molecule 1141. Therefore,
it is possible that regulatory signals are given to the
CD8+ suppressor-effector population by the CD4
+
I 1
+
a
m
t
c
l
0
30
"'k I I
ACTIVE
MS
CNS-infl
The percentages of CD4+CD45R- and CD4-CD45Rf cells
in the cerebrospinalfluid (CSF). The cross-hatched areas represent the 2 S D range from the mean, evaluated in the 37 patients
with noninflammatory neurological diseases. The triangles denote the presence of CSF pleocytosis ( > S l m d ) . The open and
closed marks (circles or triangles) represent steroid-treatedand
steroid-untreated patients, respectively. MS = multiple sclerosis;
CNS-infl = patients with inflammatory diseases in the central
nervous system other than MS.
Leu8+ suppressor-inducer subset in the CNS. Accumulating evidence has suggested that CD4 + lymphocytes lose highly expressed CD45R antigen when
activated by stimulation with phytohemagglutinin 11517}, whereas the Leu8 antigen does not disappear
permanently after prior activation {l8}. During this
process, the CD4+ lymphocytes gain high densities of
CDw29 1151 and UCHLl {l6} antigens. Since this
phenotypic change was also observed in the CD8+ cell
population { 151, the predominance of CD45R- cells
in the CSF may indicate a sequestration of onceactivated lymphocytes into the CNS in a noninflammatory condition. This assumption agrees with the report that more than 80% of lymphocytes were both
CDw29+ and UCHLl in CSF obtained from healthy
volunteers 1191. A concordant finding is that numerous T a l + cells reside in the CSF of patients with
NINDs 1203; the T a l antigen is a marker for previously activated T cells 121). It has been suggested that
lymphocytes enter the CNS through postcapillary
venules {I}, and only activated T cells can invade vas+
Matsui et al: Immunoregulation in the CNS 649
cular endothelium by degrading subendothelial extracellular matrix 122, 23). Thus, it is likely that activated T cells selectively cross the BBB and circulate
through the CNS. An alternative interpretation, however, is that the CD45R-CDw29+UCHLl+ subset
has a greater ability than CD45R+ cells to adhere to
endothelial cells 1241. It is noteworthy that CD4+
CD45R-Leu8 lymphocytes are potent producers of
interleukin-2 (IL-2) and interferon gamma upon activation 125, 261. Therefore, a relative enrichment of this
type of cell in the CSF, although small in absolute
number, seems to promote a swift immune response
to target antigens.
In the present study, the proportion of the CD4+
CD45R- cells in the CSF was significantly elevated in
the active stage of relapsing-remitting MS, irrespective
of ongoing steroid treatments. Although we classified
MS disease activity according to time from neurological relapse without examining for new MS plaques by
magnetic resonance imaging 1271, our criteria sufficed
to select patients with active MS. The increased
CD4+CD45R- cells appear to consist mainly of cells
actively entering the CNS rather than those proliferating within the CNS, since IL-2 receptor-positive T
cells have rarely been seen in the CSF or in the inflammatory lesions of MS 120, 28). By contrast, CD4CD45R+ cells were increased in the CSF of the patients with CNS-infl. A similar finding was that 2H4+
cells were numerous in tissues of viral encephalitis
C291. Thus, there may be different patterns of cellular
immune reaction in the CNS, according to the types of
CNS inflammatory conditions. Further clarification of
the specific functions of the subsets of lymphocytes
compartmentalized within the CNS could serve as an
important clue to immunoregulatory therapy for inflammatory CNS diseases.
This work was supported in part by a grant from the Ministry of
Health and Welfare of Japan, and by a grant-in-aid from the Ministry
of Education, Science, and Culture of Japan (No. 01770542).
We thank Mr S. Araya for technical assistance.
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Matsui et al: Immunoregulation in the CNS
651
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