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Decreased Lymphocyte Response To Pha Con-A And Calcium Ionophore A23187 In Patients With Ra And Sle And Reversal With Levamisole In Rheumatoid Arthritis.

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326
DECREASED LYMPHOCYTE RESPONSE
TO PHA, CON-A, AND CALCIUM
IONOPHORE (A23187) IN PATIENTS
WITH RA AND SLE, AND REVERSAL
WITH LEVAMISOLE IN RHEUMATOID
ARTHRITIS
MORTON A. SCHEINBERG, LEONILDA SANTOS, NELSON F. MENDES, and CHLOfi MUSATTI
The mechanism of poor lymphocyte transformation to mitogens was studied in selected patients with
rheumatoid arthritis and systemic lupus erythematosus.
Low lymphocyte response to PHA and Con-A in media
containing autologous and homologous sera was usually
associated with poor response to the calcium ionophore
A23187, which induces blastogenesis by a different mechanism. The low lymphocyte response to mitogens in patients
with rheumatoid arthritis could be restored by in vivo
treatment with the anthelmintic drug, levamisole. The
present findings suggest that intrinsic defects are responFrom the Disciplina de Imunologia, Departamento de Microbiologia, Imunologia e Parasitologia da Escola Paulista de Medicina and Centro de Estudos e Pesquisas Mtdicas (CEPEME), Rheumatology/Clinical Immunology, Hospital Ana Costa.
Supported by FAPESP, CAPES, CNPq and FINEP.
Dr. Scheinberg is a recipient of an investigator fellowship
from FAPESP.
Morton A. Scheinberg, M.D., Ph.D.: Research Associate,
Division of Immunology, Escola Paulista de Medicina, Head Rheumatology Clinical Immunology Unit, Hospital Ana Costa; Leonilda
Santos, Ms. Sci.: Graduate Student, Division of Immunology, Escola
Paulista de Medicina; Nelson F. Mendes, M.D., PhD.: Head, Division
of Immunology, Escola Paulista de Medicina; Chlot Musatti, Ph.D:
Assistant Professor, Division of Immunology, Escola Paulista de
Medicina.
Address reprint request to Morton A. Scheinberg M.D.
Ph.D., Disciplina de Imunologia da Escola Paulista de Medicina, Sio
Paulo, Brazil, Caixa Postal 7144.
Submitted for publication August 9, 1977; accepted in revised
form November 11, 1977.
Arthritis and Rheumatism, Vol. 21, No. 3 (April 1978)
sible for the decreased cellular response in patients with
rheumatoid arthritis and systemic lupus erythematosus.
Previous reports have indicated that patients
with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) experience decreased response of
peripheral blood lymphocytes to the plant lectins phytohemagglutinin (PHA) and concanavalin-A (Con-A)
(1-6).
The present work is concerned with the mechanism responsible for the abnormal behavior of lymphocytes from patients with RA and SLE to the mitogens
PHA and Con-A. We have attempted to demonstrate
that this low response to mitogens is independent of the
mitosis induction mechanism. Therefore, we have compared the response to PHA and Con-A with the response observed with calcium ionophore (A23 187).
A23 187 triggers mitosis by a different mechanism than
PHA or Con-A by inducing a calcium flux into the cells
without binding to the cell membrane (7). We have also
tested the possibility that the poor mitogenic response to
PHA and Con-A could be improved by in vivo administration of levamisole, an immunostimulating agent capable of altering the intracellular levels of cyclic nucleotides; these nucleotides have been shown in earlier
studies to modulate the process of lymphocyte activation (8).
DECREASED LYMPHOCYTE RESPONSE IN RA A N D SLE
P H A
>8 0
4
.
Con A
MATERIALS AND METHODS
A 23187
+
a
a
80
A
70
-a.
60
50
z
327
A
+
40
30
20
10
af.
0
Figure I. M itogenic responses of peripheral blood lymphocytes from
paiients wiili rheumatoid arthritis (triangle).systemic lupus erythemato.vus (square),arid normal controls (circles). Results are expressed as
itwan .stitnulation indexes for each group.
Patients. Twenty patients with classic seropositive RA
and 12 patients with clinically active SLE were studied. All
patients with R A and SLE met established diagnostic criteria
(9,lO). The mean age for the RA group was 45 years (range 2258) and 28 years for the SLEgroup (range 17-35). No patients
were receiving antiinflammatory or immunosupressive drugs
at the time of the study, except for some RA patients who were
kept on low dose aspirin (less than 2.0 gm a day). Patients with
SLE were considered to have active disease by established
diagnostic criteria and were all new cases without previous
therapy (1 1). The control group consisted of I3 healthy subjects ranging in age from 20 to 42 years (mean age 30).
Mitogens. The following mitogens were used: PHA-P
diluted a t 1/50, 1/100 and Con-A diluted at 1/40, 1/80 (Difco
Laboratories, Detroit, Michigan). The ionophore A23 187 was
supplied by Dr. R. Hamill of Lilly Research Laboratories
(Indiana) and used a t a dilution of 50 and 100 pg/ml.
Lymphocyte Cultures. The methods used to assay for
lymphocyte transformation are described in full elsewhere
(12). Briefly, 30 ml of heparinized venous blood were drawn,
the lymphocytes isolated on a Ficoll-Hypaque gradient and
washed three times in Hanks balanced salt solution. The cell
suspension was adjusted to 1 X 108 lymphocytes in 199 tissue
culture medium with 20% inactivated AB serum from a pool of
normal donors or with 20% heat-inactivated autologous
serum. One milliliter of this suspension was put into culture
tubes and incubated a t 37°C in 5% CO, atmosphere with 0.1
ml of either PHA or Con-A for 72 hours. The thymidine
uptake was measured after acid precipitation in a Beckman
scintillation counter. All the results were expressed as mean
stimulation index (counts per minute in stimulated culture and
counts per minute in control cultures) for each mitogen concentration. Statistical analyses were performed using the Wilcoxon's and Mann-Whitney tests.
Table I. Lymphocyte Responses to Various Concenirations of PHA. Con-A, and A23187 in Normal
Controls. RA Paiients, and SLE Patients. Results Are Expressed as Median Values and Range
Normal Controls
Mitogen
Concentration
PHA
AS*
I /50
34
( I 66-14)
NDS
PHA
1/100
Con-A
I /40
32
(58-1 1)
Con-A
I /80
ND
A23 187
50~g
ND
A23 187
* AS
t HS
4 ND
IOOrg
29
(49-16)
= Autologous sera.
= Homologous
= Not done.
sera.
HSt
39
(259- 12)
37
( I 37-12)
32
(79-7)
42
(69-12)
41
(6 1- 10)
36
(1 35-7)
Rheumatoid Arthritis
AS
HS
5
(56-1)
6
(54-1)
4
(58-1)
4
(56-1)
1
(18-1)
3
(24-1)
7
(108-1)
6
(100-1)
4
(131-1)
4
(69-1)
2
(83-1)
2
(32-1)
Systemic Lupus
AS
8
(201-2)
14
(58-1)
4
(67- I )
2
(26-1)
5
(125-1 )
3
(145-1)
HS
18
( I 13-2)
20
(65-2)
4
(79- I )
3
(31-1)
5
(68-1)
7
(88-2)
3 28
SCHEINBERG ET AL
PHA
Con A
>8 0
A23187
A
A
d
A
80
70
60
The administration of levamisole (150 mg/day
for 5 days) t o patients with RA markedly improved the
mitogen response to PHA, Con-A, and A23187 (Figure
2). Normal controls treated in a similar manner demonstrated little or no improved response, ( P < 0.01) as
shown in Figure 3. The response of lymphocytes from
SLE patients after the administration of levamisole was
not tested because there is a lack of convincing data to
support the administration of this drug to patients with
active SLE.
DISCUSSION
50
40
30
A
--P
A
20
A
10
d
A
0
+
A
In the present study the mechanism for decreased
lymphocyte response to mitogens in patients with RA
and SLE was investigated. In general, low response to
lectins was associated with unresponsiveness to the ionophore A23 187. Administration of levamisole restored
the mitogenic response to values comparable to those
obtained in the control group.
cA
P H A
Con A
A23187
L i >8 0
Figure 2. Stittiulation indexes o/’ peripheral blood 1jvnphocyre.q frottr
paiienrs with rheumatoid arthritis before (dark triangle) and alter (lighr
rriungle) rhe admitrisiratiorr of Ieuanrisole.
RESULTS
In most of the experiments described, there was a
close approximation between the counts per minute of
unstimulated lymphocytes for a given group of patients
and for normal controls; therefore, the differences observed in the stimulation indexes between the various
groups are consequences of a difference in the incorporation of tritiated thymidine by the stimulated lymphocytes.
As shown in Figure 1 and Table I , there was a
significant impairment in the incorporation of tritiated
thymidine by lymphocytes from patients with RA and
SLE ( P < 0.01 and P < 0.05, respectively). Except for
an occasional patient, the response of peripheral blood
lymphocytes from patients with RA and SLE to the
ionosphere A23187 were similar to that observed for
PHA and Con-A.
The differences in the lymphocyte response of the
RA and SLE patients and controls were not reversed by
culturing the lymphocytes in normal homologous sera
(Table I).
80
70
0
60
0
50
0
40
0
0
0
+
30
20
0 0-u0
0
0
0
10
0
Figure 3. Srittrularion indexes 01peripheral blood lymphocyres from
riorttral ooluntrers byfiwe (dark circle) and after (light circle) the adniinisrrariotr u/’ leoaniisole.
DECREASED LYMPHOCYTE RESPONSE IN RA A N D SLE
Lymphocyte activation by mitogens apparently
depends on a calcium influx via endogenous calcium
ionophore that acts as a signal for cyclic nucleotides to
trigger cellular proliferation (13). Therefore agents that
increase cellular levels of cyclic guanosine monophosphate (GM P) promote the proliferative and secretory
functions of lymphocytes, and those agents that increase cyclic adenosine monophosphate (AMP) act to
decrease such functions. The transfer of calcium into
lymphocytes by A23 187 bypasses physiologic membrane
events, resulting in proliferation of normal human lymphocytes. Levamisole, a derivative of imidazole, is an
agent that enhances the catabolism of cyclic A M P and
inhibits the catabolism of cyclic GMP, which is implicated in the process of lymphocyte activation.
The inability of lymphocytes from patients with
R A and SLE to respond to A23187 and the increased
mitogenic response after the administration of levamisole are compatible with the theory that a defect at the
calcium fiu~-cyclicnucleotide step might be responsible
for the poor mitogenic response in these patients. In
addition, the fact that no differences were observed in
the response of lymphocytes cultured in autologous sera
seems to rule out the possibility that a circulating humoral factor is responsible for the present findings.
The specificity of our findings comparing rheumatoid arthritis patients to patients with nonimmune
chronic disorders cannot be solved at the present time,
because preliminary investigations testing the effect of
levamisole in these patients gave variable results (14).
The levamisole-associated improved mitogenic response
observed in this study could be related to the beneficial
effects of this drug which have been observed in some
patients with rheumatoid arthritis (15, 16).
REFERENCES
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ACKNOWLEDGMENTS
We would like to thank Mrs. Anna Freund for secretarial assistance and Dr. Ian McKay for the critical review of
the manuscript.
329
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patients, reversal, con, sle, levamisole, ionophore, decrease, a23187, response, pha, arthritis, calcium, lymphocytes, rheumatoid
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