Distribution of Cells expressing
12
ABSTRACT
Rheumatoid Arthritis (RA) ditandai dengan peradangan kronis, infiltarsi sel-sel,
seperti monosit, limfosit dan proliferasi jaringan ikat. Fc reseptor terhadap
IgG(FcyR) pada makrofag, granulosit dan limfosit memperlihatkan peranan yang
penting pada sel-sel stimulating efektor melalui reseptor ini.
Kita telah melakukan penelitian terhadap distribusi sel-sel permukaan FcyR
dari 12 jaringan sinovial pasien RA dan 3 orang pasien OA post operasi dengan
menggunakan monoklonal anti bodi CD 14, CD 64, CDw32 dan CD 16 pada
setiap cryostat section melalui metoda pewarnaan immunohistologi.
Densitas sel dan derajat infiltrasi limfosit memperlihatkan nilai yang signifikan
tinggi pada Ra di banding OA. Jumlah sel-sel yang mengekspresikan FcyRI,
FcYRII, dan FcyRIII di hitung sebagai persentase monosit/makrofag. Range nilai
berkisar antara 18 – 61 dan tidak ada perbedaan yang signifikan ditemukan antara
RA dan OA, dan dalam hal ini tidak ada hubungan dengan derajat infiltrasi
limfosit.
Kita mengamati pola ketiga Fc reseptor, yang menunjukkan perbedaan
regulasi dan ekspresi dari molekul ini pada sub populasi yang di pandang
mempunyai fungsi yang penting.
kata kunci; Rheumatoid Arthritis, Fc Receptors, monoclonal anti body
INTRODUCTION
Rheumatoid arhtritis (RA) is a chronic
inflammation disease that affect synovial
tissue (ST) in multiple joints. It is
associated with marked inflammatory
reaction ivolving the synovial fluid,
synovial membranes, and periarticular
tissues. This recation is characterized by
infiltration of the synovium with
mononuclear
cells
(MNC),
and
accumulation
of
leucocytes
predominantly neutrophils (PMN).(1.2)
The general features of synovitis
are common to most inflammatory
arthropies, the histological features are
increased lining layer cells depth, thougt
tobe
due the influx
of type A
macrophage
derived
lining
cells
associated with local proliveration pf
type B fibroblast–like synovities. The
sub lining layer is characterized by a
mixed
inflammatory
infiltrate
consisting mainly of macrophages and T
lymphocytes. Dendritic cells which are
spesialized
antigen presenting cells
althougt to be important
in the
stimulation of immune responses, are
found in this layer, particularly in the
perivascular areas. Other cells found
include B lymphocytes and plasma cells,
mast cells and some nethrophils.(3)
The inflammmatory changes in the
synovial membranes of patients with
osteoarthritis
(OA)
are
almost
indistinguishable from those seen in
patients with an inflammatory infiltrate,
particularly in the end stage sof disease
investigated during joint replacement.
However, the cellular infiltrat is
generally less significant than in the
inflammatory arthropies.(4)
Monocytes or macrophages are an
important component of inflammatory
lessions particularly those of a chronic
nature, in RA it has veen shown that
Majalah Kedokteran Andalas Vol.22. No. 1. Januari – Juni 1998
Distribution of Cells expressing
they are only cells type whose number
correlated with the degree of joint
erosion. Cytokines are released from
activated cells and macrophages have
been implicated as mediators of both
inflammation and joint destruction.(2.5)
Cellular
Fc
receptoprs
and
complement receptors are molecules
that mediated between in the innate and
adaptive arms of the immune system
and the are important molecules for the
effector punctions of macrophages.
Obviously,
macrophages
and
complement belong to the innate arm of
the immune system, while antibodies are
produced bt B cells of the adaptive
immune system. Fc receptors cand bind
Fc portion of particularly antibody
subclasses. Three separates FcR for IgG
(IgG is mostly antibody subclasses in
RA) have been identified in macrophages
which are specific for different IgG.
These receptors are also designated
FcRI, FcRII, and FcRIII (fig I). FcRI
and FcRII can be independently
modulated when macrophages are
attached to surface coated with the
appropriate sub class of antibody. The
FcRIII molecule of macrophages
mediates internalization pf antibody –
coated particles.(6)
The pathogenesis of RA remain
unknown despite advances in the
understanding of disease mechanism.
One hypothesis proposes that RA is a T
lymphocyte–mediated
respon
to
unidentified antigen. Other have
emphasized the relative importance of
macrophages abd fibroblast in the
rheumatoid arhtritis.(5)
The aim of present study was
to
examine the expression of surface
molecules
on
synovial
tissue
13
macrophages and sequestered PMN in
RA and OA to ascertain whether
different in expression might play a role
of inflammatory and their subsequent
activation state.
MATERIAL AND METHODE
Synovial tissue
Synovial tissue samples were obtain
patients with active rheumatoid arhtritis
(RA) and osteoarthritis (OA) while
undergoing
arthroplasty
or
synoviectomy in hospital. RA and OA
were diagnosed according to the criteria
of
the
American
Rheumatism
Association (ARA).
Synovial tissue was immediatelly
processed
for frozen sections cell
preparation
Cells
Mononuclear cells were separeted
from synovial tissue and isolated by
density gradient centrifugation over Fical
Hypaque (Pharmacia fine Chemical,
sweden). The cells were washed, and 3 x
106 cells were counted for preparation
on the slide, then air dried and fixed to
examine
by immunohistochemistry
staining methods.
Monoclonal antibodies
The following monoclonal antibodies
(Mab) (behring werke AG, Marburg)
were used :
MabCD16 (direct against FcRI)
Mab CDw32 (Direct against FcRII)
Mab CD 64 (Direct against FcRIII)
Mab CD14 (Direct against macrophages)
Immunoprexidase technique
Cells preparations or section were
incubated serially in hummidified
Majalah Kedokteran Andalas Vol.22. No. 1. Januari – Juni 1998
Distribution of Cells expressing
14
Receptor
FceRI
FcRII (CD32)
FcRIII(CD16) FcRI (CD64)
Structure
 45 kDa
 40 kDa
 50-70 kDa
 33 kDa
 9 kDa
Bindin
g order
of
affinit
y
Cells
type
IgE, 1010 M-1
Mast cells
74 kDa
FceRII
(CD23)
 45 kDa

IgG1,2x106M-1
1)IgG1
2)IgG3= IgG4
3)IgG2
Macrophages
Neutrophils
Eusinophils
Platelets
B cells
IgG1,5x105M1
IgG1 = IgG3
IgG1,106M-1
1)IgG1
2)IgG3
=
IgG4
3)IgG2
Natural
Killer Macrophages
Cells
Neutrophils
Neutrophils
Eusinophils
Eusinophils
Macrophages
IgE
affinity
low,
Eusinophils
Activated
B
cells
Follicular
dendritic cells
Figure 1 : Distinct receptor for the Fc region of the different immunoglobulin isotypes
(antibody subclass) are express on differenet accesory cells.
chamber, primary Mab (mouse
antihuman) diluted in fresh phosfat
buffered saline (PBS Sigma) for 30
minutes, and biotinylated anti mouse
secondary antibodies followed by
horseradish
peroxidase–conyugated
evidin biotyn complexs for 30 minutes
each (Sigma, St. Louis Roma, MO).
With
five minutes PBS washes
between steps. Collor wash develoed
which 3.3 diaminobenzidine and 1%
hidrogen peroxidase 1% for ten
menutes. Cells or section were
counterstained with hematoksilin
dehydrated and coverslip.
Statistical analysis
Result are expressed as the mean 
SD of the mean. Student t’test and
mann Whitney U test were used to
campare the results. P < 0.05 was
considered significant.
RESULTS
Sinovial tissues from twelve RA
patients (9 female and 3 male) and 3
OA patients (1 female and 2 male) of
mean 56.8 years (range 39 – 61), were
studied. All of RA patients had severe
disease with Larson grade III – IV, 8
of them were RF positive.
Comparison of lymphocytes cells
number on synovial tissues of RA
patients with OA patients is
summaryzed in table 1. The
Majalah Kedokteran Andalas Vol.22. No. 1. Januari – Juni 1998
Distribution of Cells expressing
15
Table 1. Mean percentages of lymphocyte of synovial tissue cells from
RA patients compared with OA patients.
RA (n = 12)
OA (n = 3)
P
Lymphocytes
0.04*
70.3  3.5
40.3  4.3
Value are means  SD
* Statisticaly significant
Limphocytes cells count in RA
patients was significantly higher than
in OA (p < 0.05).
Expression of each the marker was
compared on munonuclear cells from
the RA and OA samples with the range
from 18 to 61% as seen in table 2.
There were no significant defferent
between tha mean percentages of
CD16, CDw32 and CD64 in RA and
OA (p > 0.05). The results from
subset CDw32 and CD64 were also
no significant difference. But the mean
percentage of CD16 was significantly
lower than the other (p < 0.05
In contrast, with CD14 expression,
these
mean
percentages
was
significantly higher in RA than OA
(p< 0.05).
The pattern of expression of
differen markers are showed in
figure 1.2 and 3. CD14 + cells were
predominantly in RA, their distribution
within).
sinovium were mainly
localized to the deeper linning layer
(fig. 1). The exprssion of CD 16 and
CDw32 were less nomerous than
CD14. The distribution of CD 64
mainly in the superficial (fig.2). The
count and distribution of positive cells
with CD 16 in RA and OA were
similar (fig. 3).
DISCUSSION
A finding in this study was that
significantly fewer sinovial cells
expressed CD14 in OA compared
with RA. In contrast expression of
FcR were similar in amount and
distribution suggest that there are
important fenotipe of macrophage
may play important role in articular
destruction.
Based on the relationship between
macrophages and articular demage,
this study was undertaken using Mab
which recognized anti gen expressed
by activated or mature macrophages
(CD 14). The observation emerged
wich may provide important insight
into the pathogenesis of articular
destuction. Synovial macrophages
alone correlated with the radiologic
course and outcome of RA, and
Table 2. Mean percentages of mononuclear cells (MNC) isolated from synovial
tissues of RA patients and OA Patients expressing CD64, CDw32, Cd16 and
CD14 antigens
MoAB
RA (n = 12)
OA (n = 3)
CD16 (FcRI)
CD32 (FcRII)
CD64(FcRIII)
CD14
22.7  4.6
35.8  8.3
35.1  13.3
20.6  8
20.7  2.5
35  7
34.7  8.1
14.7  11.6
Majalah Kedokteran Andalas Vol.22. No. 1. Januari – Juni 1998
P
0.5
0.9
0.0
0.07*
Distribution of Cells expressing
lining layer macrophages differed in
phenotype from those in the sublining
layer, supporting the suggestion that
they may represent and important
subpopulation
of
synovial
macrophages with a role in articular
destruction.
Many
destructive
mediators produced by these cells.
Some mediators, such as collagenase
and interleukin-1, are clearly produced
by synovium macrophage. There is
good evidence that many other also
arise mainly from lining layer cells. At
the cartilago-pannus junction, were
pannus lead to joint destruction,
abundant macrophages and synovium
fibroblast
are
evident,
and
macrophage–derived cytokines have
been demonstrated.(8)
The
glycoylphosphatidylinositol
(GPI)linked FcRIII (CD16) is the
domininant Fc receptors for activation
via physiological immune complexes
in PMN and is released upon in vitro
activation of PMN. The expression of
FcR, FcRII, and FcRIII were
similar on both RA and OA, but the
expression of FcRI was lower than
FcRII and FcRIII. Althoug antigen
shedding may explain the decrease of
CD16 on tissue PMN, the regulatory
mechanism in vivo are likely to be
more complex. There are different in
phenotype and also different of cells
may lead chronic inflammatory
synovium.
The
central
pathology
of
Rheumatoid synovitis is the interaction
of T lymphocyte cells and synovial
cells, which drives the production of
cytokines. Significant number of
activated T lymphocyte are detected in
16
inflammatory synovium in RA. The
accummulation of T lymphocyte in
RA synovium represent 2 distinct but
closely related functions of T
lymphocyte mobility: recirculation and
recognition. The former is T cell
interaction with endothelium, which is
important
to cell entry
into
inflammed synovium., and the later is
the interaction of T lymphocyte to
opposing cells such as antigent
presenting cells or fibroblast which
leads to stimulation of oppsing
partner
cells as well as
T
lymphocytes. Immunohistochemical
analysis shows that activated T
lymphocyte expressed strongly in
active RA. However, in OA synovium
this expression is different (9).
Synovial microscopic change in
OA was considerable variable in the
thickness of the synovial lining layer.
Most linning layer appeared to have
cells continuity, even in the normal
synovium, but were present in 37%
severe grades of OA synovial
membranes, often resembling the
synovial membranes from patients
with RA.(1)
CONCLUSION
In RA patients an increased a
number of synovial tissue lymphocyte
may caused
by chronic antigen
stimulation. The expression of FcRI,
FcRII and FcRIII on the cells is
regulated accordinf to the state
macrophage activation. The distintive
ataining patterns for three
Fc
receptors, seggesting, that there maybe
differential regulation and expression
of these molecules on subpopulation
Majalah Kedokteran Andalas Vol.22. No. 1. Januari – Juni 1998
Distribution of Cells expressing
which might
importance
be
of
17
KEPUSTAKAAN
1.
2.
45Rbdim, CD27-memory T cells in
the peripheral .
functional
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