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Drugs affecting the release of rheumatoid factor in a plaque-forming cell assay.

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114
DRUGS AFFECTING THE RELEASE
OF RHEUMATOID FACTOR IN A
PLAQUE-FORMING CELL ASSAY
TERRY L. MOORE, DICK L. ROBBINS, JEAN E. ROSE, and JOHN H. VAUGHAN
Addition of propranolol to the agarose phase of a
plaque-forming cell (PFC) assay for rheumatoid factor
(RF) caused reduction in the number of plaques seen. This
reduction in rheumatoid factor plaque-forming cell
( R F PFC) did not depend upon an effect at the 8-adrenergic receptor, since d- and I-propranolol reduced equally
well. Furthermore, in a series of polycyclic compounds
with varying &receptor blocking capabilities there was no
agreement between plaque reduction and blocking. When
propranolol was tested in the agarose in an anti-sheep
erythrocyte (SRC) plaque assay (anti-SRC PFC), it had
no inhibitory effect, but it was capable of inhibiting the
generation of new anti-SRC PFC in an in vitro culture.
Propranolol is thought to exert these effects through its
membrane stabilizing (anesthetic) properties.
From the Division of Rheumatology, Department of Clinical
Research, Scripps Clinic and Research Foundation, La Jolla, California 92037.
Supported by research grants AM 07144, CA 14126 and RR
00833 from the National Institutes of Health, and by a grant from
the Kroc Foundation.
Terry L. Moore, M.D.: Assistant Professor, Department of
Medicine, St. Louis University, St. Louis, Missouri 63103; Dick L.
Robbins, M.D.: Assistant Professor of Medicine, Section of Rheumatology TB 171, University of California at Davis, Davis, California
95616; Jean E. Rose: Division of Rheumatology, Scripps Clinic &
Research Foundation, La Jolla, California 92307; John H.Vaughn,
M.D.: Chairman, Department of Clinical Research Scripps Clinic &
Research Foundation, La Jolla, California 92307.
Address reprint requests to Dr. John H. Vaughn, Chairman,
Department of Clinical Research, Scripps Clinic & Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92307.
Submitted for publication June 9, 1977; accepted August 10,
1977.
Arthritis and Rheumatism, Vol. 21, No. 1 (January-February 1978)
The production and release of rheumatoid factor
(RF) by lymphoid cells of certain patients with rheumatoid arthritis (RA) have been shown in bone marrows,
synovial fluids, and peripheral bloods by a hemolytic
plaque-forming cell ( R F PFC) assay (1). The target cells
used were sheep cells sensitized with reduced and alkylated rabbit IgG antibody, which lysed directly in the
presence of added rheumatoid factor plus complement.
Dependence of the R F PFC on protein synthesis and
intact microtubular excretory system was indicated by
studies in which cycloheximide or vinblastine was added
to the agar. In other studies the addition of propranolol
also markedly reduced the number of R F PFC observed. This effect of propranolol did not seem to be
explained on the basis of its ability to block 6-adrenergic
receptors. This study was designed to learn more about
the propranolol effect.
MATERIALS AND METHODS
Subjects. The human lymphocytes studied were from
normal laboratory personnel or from patients with seropositive RA, high numbers of R F PFC in their blood, and
erythrocyte sedimentation rates greater than 40 m m per hour.
Fifty to 250 ml of each patient’s blood were drawn into
heparinized syringes and allowed to sediment for an hour. The
leukocyte-rich plasma and top 10-20% of red cells were expressed into separate tubes and the remaining red cells then
returned to the patients by intravenous infusion.
Preparation of Lymphocytes from Patients. The leukocyte-red cell suspension was diluted 1 : 2 in normal saline and
layered in 30 ml aliquots over 12 ml of ficoll-hypaque (2).
RELEASE OF R F
After centrifugation at 1200 rpm for 30 minutes at room
temperature, the lymphocyte layer was aspirated and washed
once by centrifugation in balanced salt solution (BSS). It was
then washed twice more in Autopow (Flow Labs) fortified
with 10% fetal calf serum (FCS), 1.0% sodium bicarbonate,
0.4% sodium pyruvate, 0.4% nonessential amino acids, and
0.4% 1-glutamine at 4OC for 10 minutes at 1200 rpm. The cells
were then resuspended in FCS, counted, and used for R F PFC
assay.
Sensitized Sheep Cells. Rabbit IgG anti-sheep hemolysin (BBL, Division of Bioquest) was separated by Sephadex
G-200 gel filtration or DE-52 chromatography. It was reduced
for 20 minutes at room temperature by 0.0045 M dithiothreitol, alkylated with 0.009 M iodoacetamide, and dialyzed overnight in phosphate buffered saline (PBS), pH 7.4. To allow
greater sensitization of the sheep cells without troublesome
agglutination, the reduced and alkylated hemolysin was
treated in the manner described by Nisonoff and Palmer (3)
for making hybrid molecules with only one antigen-reactive
group. The hemolysin was brought to pH 2.8 with 0.5 M
glycine-HCI buffer for 60 minutes at room temperature, then
allowed to dialyze back to neutrality overnight with PBS, pH
7.4. Before this treatment the OD,, agglutination titer, and
Coombs titer of the IgG hemolysin were 2.680, 1: 1024, and
1:1024. After treatment the values were 1.004, 1:32, and
1 : 512.
Four-tenths milliliter of the reduced, alkylated, and
acid-treated rabbit IgG hemolysin was diluted in 9.6 ml of
BSS; to this was added an equal volume of a 2% sheep red cell
(SRC) suspension in BSS. The suspension was incubated 25
minutes at 37OC, then centrifuged once for 10 minutes at 1200
rpm at 4°C. The sensitized SRC were resuspended to 6.7% in
BSS.
Source of Complement. Lyophilized guinea pig serum
(Hyland Labs) was reconstituted with the diluent provided.
PFC Assay for RF Production (1). Five-tenths milliliter
of 0.5% agar (Agarose A-37 Induboise) in Autopow at 44°C
was mixed with 0.05 ml of a 6.7% suspension of sensitized SRC
and 0.1 ml of lymphocyte suspension containing 1-5 X 10"
lymphocytes. Compounds being tested for inhibitory capabilities were added to the melted agar in 0.05 ml volumes in the
appropriate concentrations. The cell-agar mixture was quickly
poured onto precoated microscopic slides and allowed to
harden. The slides were incubated at 37°C for 90 minutes in a
moist chamber, then immersed in guinea pig complement diluted 1 : 10 in BSS, and incubated an additional 90 minutes at
37°C. R F PFC were enumerated under magnification. Control
slides containing nonsensitized SRC were invariably negative,
indicating the anti-IgG specificity of the R F PFC examined.
Plaque formation was complement-dependent since no
plaques formed when complement was omitted from the system. Replicates of three to four slides were always set up, the
reported values were the means f standard error (SE).
The viability of the lymphocytes in Autopow containing inhibiting drugs was tested with trypan blue. This revealed
> 95% viability throughout the incubation period. Furthermore, LDH values of supernatants of the lymphocytes incubated in the inhibitors were not significantly different from
control lymphocytes without inhibitor, indicating no escape of
this cytoplasmic enzyme.
Chemicals Tested for Inhibitor Activity. Dibutyryl cy-
115
clic AMP was obtained from Calbiochem. 8-Bromo-cyclic
GMP, isoproterenol, carbamylcholine, and quinidine were
from Sigma. Atropine was from Eli Lilly and Co. D, I-propranolol, d-propranolol, I-propranolol, and practolol were
from Ayerst Laboratories. Butoxamine was from Burroughs
Wellcome Co. The following compounds were obtained from
Dr. Robert Meyer (4) at Parke, Davis & Co:
Compound
Abbreviation
a-[(tert-butylamino) methyl]6-methyl-2 quindineethanol
Meth-quin-eth
[6)*
(t-but-meth)
a-[(tert-butylamino) methyl]6-phenanthridineethanol
2
HCI
I-(tert-butylamino)-3-(Cphenanthridinyl)-2-propanone,
monooxalate
Phenanth-eth
[ 161
(t-but-meth)
I-benzyl-a-[(tert-butylamino)
methyl]-2-benzimidazoleethanol
a-[(tert-butylamino) methyl]1 -2-benzimidazoleethanol
Phenanth-ProP (t-but) 17
Benzyl-benzimid-eth (t-butmeth) [I91
Benzimid-eth
[211
(t-but-meth
Brackets refer to number of compound in reference 4.
Human Lymphacyte Cultures (5). Two to 5 X 10" lyrn
phocytes separated by ficoll-hypaque were cultured in 2.0 ml
of supplemented RPMI-1640 and 10% human AB serum preabsorbed to remove natural anti-SRC antibodies and in I :500
pokeweed mitogen (Grand Island Biologicals). Cultures were
maintained 7 days in 5% CO, and air at 37°C. Cells were
harvested by centrifugation, washed once with RPMI i n 1%
FCS, and plaqued as above.
RESULTS
In our original studies we were interested i n the
possibility that R F release by lymphocytes in the agarose phase of the assay was modulated by cyclic nucleotides. To test this, we used lipid soluble cyclic nucleotides and a variety of agonists and antagonists of the
receptors controlling cyclic nucleotide levels. These included dibutyryl cyclic AMP, 8-bromo-cyclic GMP, d , Ipropranolol, carbamylcholine, and atropine. Of these,
only d, I-propranolol had any effect. A t lo-' M ,propranolol completely suppressed RF PFC. The inhibition
was not reversed by an equimolar amount of isoprotereno1 (Figure 1 ).
It seemed likely that the inhibition by d , l-propranolol was not mediated through its @-adrenergic
blocking effects, since the @-stimulator isoproterenol did
not reverse the propranolol effect and since no corresponding or reciprocal effects were seen with the other
additives tested. W e suspected that d , I-propranolol ex-
MOORE ET AL
116
s
0
Y
4
control
1 0 - 4 -5 -6
10-4 -5 - 6
10-4 -4 -4 - 4 -4
d-, 1-,
propranolol
d-, 1-,
propranolol
10.4 - 4 -4
I A C di 8B
isoproterenol
Figure 1. Effectsof agonists and antagonists of adrenergic and cholinergic receptors. and of cyclic nucleotides,
on numbers of rheumatoid factor-producing lymphocytes ( R F PFC) in a hemolytic plaque assay. Test
substances were added to the agarose simultaneously with the lymphocytes. Slides were then incubated 90
minutes at 37'C followed by immersion in complement for another 90 minutes at 37°C. I = isoproterenol; A
= atropine; C = carbamycholine; d i = dibut cyAMP; 8B = 8-Br-cyGMP.
T
0
c
L
E
0
0
.c
0
0
LL
4
LL
a
10 5 2.5
(x~O-~M)
Control
1-propranolol
10 5 2.5
1 0 - 4 -5 -6
10-4
1x1 0-5MJ
d-propranolol
practolol
butoxamine
Figure 2. Lack of correlation of inhibition of RF PFC with 0-adrenergic blockade. d-Propranolol has no 0blocking effect. Practolol and butoxamine have /3, and b2 blocking effects, respectively. Conditions of the
experiment were like those in Figure I .
RELEASE OF RF
117
Table 1. Independence of Inhibition of RF PFC from @-Receptor
Blocking Activity
Agent
Phenanth-eth (t-but-meth)
Phenanth-prop (t-but)
I-propranolol
Meth-quin-eth (t-but-meth)
Benzimid-eth (t-but-meth)
d-propranolol
Benzyl-benzirnid (t-but-meth)
Quinidine
Relative
@-Receptor
Blocking (4)
930
382
I00
2
I
1
0
0
RF PFC
Inhibition
+++
+
+++
+
++
+++
+++
++
erted its effect through its membrane stabilizing (anesthetic) property (6). To test this we compared the effects
of d-propranolol, I-propranolol, butoxamine, and practolol. D-propranolol has membrane stabilizing properties but essentially no &blocking activity. Practolol and
butoxamine have no membrane stabilizing properties,
but are PI and @,-blockers. L-propranolol has both effects. At lo-' M , both d-propranolol and I-propranolol
reduced the numbers of R F PFC by more than 90%; at 5
X
M there was 30-8096 reduction (Figure 2). Practolol and butoxamine in the same concentrations had no
significant effects.
These observations supported our suspicion that
the inhibitory effect of d, I-propranolol on RF release
occurred through its membrane stabilizing action.
Membrane stabilizing activity in a number of P-blockers
studied by Zaagsma and Nauta (7) was found to be
enhanced by having a second phenyl ring present in
polycyclic (naphthyl) form. Propranolol has a naphthyl
ring structure, whereas practolol and butoxamine have
only single rings.
A further set of polycyclic agents demonstrated
by Meyer et ul. (4) to have varying P-blocking activities
was investigated for their effects on R F PFC (Table 1).
All agents were tested at lo-' and
M . Phenanth-eth
(t-but-meth) and benzyl-benzimid-eth (t-but-meth)
markedly inhibited PFC numbers at lo-' M . Benzimideth (t-but-meth) had moderate inhibition. Phenanthprop (t-but) and meth-quin-eth (t-but-meth) produced
minimal or no inhibition. Thus among these compounds, inhibition of RF PFC again did not correlate
with P-blocking activity.
None of the above compounds were anticomplementary because they induced no change in hemolytic titer of a standard R F in guinea pig complement
or in the shape of the curve.
We then tested the effects of propranolol on an
anti-SRC PFC system to determine whether the propranolol effect would be seen with a more standard
antibody than RF. Human anti-SRC PFC were stimulated by pokeweed mitogen, as described by Fauci and
Pratt ( 5 ) . Anti-SRC PFC produced in this manner were
not susceptible to inhibition by lo-' M propranolol
(right side, Figure 3). Thus although propranolol was
capable of inhibiting the release of an autoantibody
(RF) from human lymphocytes, it was not capable of
inhibiting the release of antibody to a foreign antigen
(SRC).
To test whether propranolol, though incapable of
suppressing anti-SRC release, could nevertheless have
immunosuppressive effects if added at an earlier stage in
antibody development, propranolol was added to the
Fauci-Pratt ( 5 ) culture system at the initiation of culture
and the number of PFC generated in 7 days determined.
Propranolol markedly inhibited the development of
anti-SRC PFC at lo-' M and partially inhibited at 2.5
and 5.0 X
M . The results are shown on the left side
,O01
Ce 1 5 0 1
-
Propranolol
Present
During C u l t u r e
Propranolol Added
a t End o f Culture
10 5 2.5
10 5 2.5
0
Control
[~10'~M)
(x~O-~M]
Concentration d-propranolol
Figure 3. Effects of propranolol on anti-SRC PFC generated from human peripheral blood lymphocytes cultured 7 days in pokeweed mitogen.
Propranolol failed to inhibit anii-SRC PFC when added only to agarose
in the final assay (right). (Conditions were like those in Figure I . )
Immunosuppression occurred when it was added to the lymphocytes at
the initiation of culture. The viabilities of the cells by trypan blue
exclusion were 60-70% in all propranolol tubes and were equal to those
in the control tubes. Total numbers of recovered cells in propranolol at
10 X 1 O F M were 65% thai of the controls. At 5 and2.5 X
M propranolol the recovery was equal to the controls.
MOORE ET AL
118
of Figure 3. Practolol and butoxamine failed to inhibit
both culture systems at all concentrations.
Thus propranolol may inhibit anti SRC production if added early to the cultures, although it does not
inhibit anti-SRC release when added only at the end,
during the final assay in the agarose. The inhibition
occurred at propranolol concentrations that were nontoxic by the criterion of trypan blue staining. The low
cell recovery at the highest propranolol concentration
(see legend, Figure 3) may have been caused by a cytotoxicity not revealed by trypan blue exclusion, or possibly by inhibition of cellular reduplication by otherwise
nontoxic membrane effects of the drug.
I n one experiment RF PFC has been generated in
culture using the Fauci-Pratt conditions. Propranolol
completely inhibited at 10 X lo-&M and partially inhibited at 5 X lo-’ M.
DISCUSSION
The experiments that led to the present study
initially were intended to investigate the effect of cyclic
nucleotides on RF PFC. Melmon et al. (8) had suggested that in splenic leukocytes from immunized mice
cyclic AMP may suppress plaque formation, as do drugs
that stimulate increased levels of cyclic AMP or decrease
its degradation. I n our RF PFC system, however,
dibutyryl cyclic AMP and 8-bromo-cyclic GMP showed
no effect on numbers of PFC and thus no effect on RF
release (I). Nor did drugs that are known to increase or
decrease cyclic AMP or cyclic GMP levels through actions at 8-adrenergic and cholinergic receptors have an
effect. This is consistent with the claim of Niaudet et al.
( 9 ) that human B cells lack @-adrenergicreceptors. On
the other hand, d, I-propranolol did markedly inhibit
the numbers of RF PFC.
The inhibitory effect of propranolol on RF release did not correlate with @-adrenergicblocking activity. D-propranolol which is devoid of 8-adrenergic
blocking activity but has membrane stabilizing effects
showed inhibition; practolol and butoxamine which
have full @-blockingactivities but almost no membrane
stabilizing effect did not. Differences such as these were
also seen with the polyclonic compounds studied in
Table 1. I n studies to be reported separately, propranolol has been shown to interfere with patching and
capping on B lymphocytes (10). We suspect, therefore,
that RF release from the lymphoid cells of patients with
RA is also related to stabilization of the cell membrane.
Our prior studies ( I ) on RF PFC have indicated
that ongoing protein synthesis is needed for their full
expression, since the numbers of RF PFC were significantly reduced by the inhibitors of protein synthesis
cycloheximide and puromycin at 10 pg/ml. By contrast,
anti-SRC PFC from mice immunized 5 days previously
were not reduced by cycloheximide ( I I ) . We interpreted
this to mean that in the anti-SRC there is a more rapid
rate of antibody production than in RF PFC, with correspondingly larger amounts of “storage” antibody in
the cytoplasms of the anti-SRC PFC. We suspect that
such differences in rates of antibody synthesis or in
quantities of stored antibody may also be responsible in
some way for the differential susceptibility of RF PFC
to propranolol.
Although propranolol did not show any ability
to inhibit the release of antibody from anti-SRC PFC, it
did show an in vitro immunosuppressive effect against
the generation of anti-SRC PFC in culture when it was
added to the lymphocytes at the initiation of the culture.
So far we have been able to conduct only a single experiment on the generation of RF PFC in vitro, but in this
one instance the results were comparable. Whether
propranolol may be a useful immunosuppressive agent
for human autoimmune disease is not yet known. At the
very least, larger doses than those in common use today
would probably be needed, if concentrations at
M
or more were to be reached in the blood. Alternatively,
other membrane active drugs derived from propranolol
but with higher potency may be developed.
REFERENCES
I . Vaughan JH.Chihara T, Moore TL, et al: Rheumatoid
factor-producing cells detected by direct hemolytic plaque
assay. J Clin Invest 58:933-941,1976
2. Boyum A: Separation of leukocytes from blood and bone
marrow. Scand J Clin Lab Invest 21 (suppl 97):77-89,
1968
3. Nisonoff A. Palmer JL: Hybridization of half molecules of
rabbit gamma globulin. Science 143:376-379, 1964
4. Meyer RF, Stratton CD, Hastings SG,et al: 8-adrenergic
blocking agents. Nitrogen heteroaryl-substituted 2-propanolamines and ethanolamines. J Med Chem
16:1113-1I16, 1973
5. Fauci AS, Pratt KR: Activation of human B lymphocytes.
J Exp Med 144:674-684, 1976.
6. Nickerson M, Collier B: 8-adrenergic blocking agents,
The Pharmacological Basis of Therapeutics. Fifth edition.
Edited by LS Goodman, A Gilman. New York, MacMilIan Publishing Co, 1975, pp 547-552
7. Zaagsma J, Nauta W Th:In vitro 8-adrenergic blocking,
anti-arrhythmic and local anesthetic activities of a new
RELEASE OF R F
series of aromatic bis (2-hydroxy-3-isopropylaminopropyl) ethers. J Med Chem 17:507-513, 1974
8. Melmon KL, Bourne HR, Weinstein Y: Hemolytic plaque
formation by leukocytes in vitro. J Clin Invest 53:13-21,
1974
9. Niaudet P, Beaurain G, Bach MA: Differences in effect of
isoproterenol stimulation o n levels of cyclic A M P in hu-
1 I9
man B and T lymphocytes. Europ J Immunol 6:834-836,
I976
10. Dunne JV, Peters CJ, Moore TL, et al: The effects of
propranolol on rosetting, mitogenic, and capping formations of lymphocytes. In preparation
1 1 . Moore TL, Rose J, Vaughan JH: Inhibition of mouse
anti-sheep erythrocyte plaque-forming cells. Unpublished
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