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Differential effects of propranolol on lymphocyte rosette formation and response to plant mitogens.

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The effects of propranolol on various lymphocyte
functions were studied to gain a better understanding of
the recently demonstrated suppressive effect of propranolo1 on rheumatoid factor production. D- and L-propranolol
at a concentration of 1 X lO-'M inhibited the formation
of human EA rosettes. The inhibition occurred within one
minute of adding the compounds, was reversible, and did
not affect cell viability. Addition of propranolol to preformed EA rosettes failed to disaggregate them. Patching
and capping of SIg by an Fab'2 anti-IgG were inhibited at
2.5 X 10-5M and above. Propranolol at 2.5 X 10-6M also
inhibited lymphocyte response to phytohemagglutinin and
pokeweed mitogen without evidence of cell toxicity by
trypan blue staining or absolute numbers of surviving cells.
Congeners of propranolol with mainly beta adrenergic
blocking properties did not show inhibitory effects. The
inhibitory activities of propranolol are interpreted in terms
From the Division of Rheumatology, Department of Clinical
Research, Research Institute of Scripps Clinic, La Jolla, California.
Supported by N I H grants CA 14126, AM 07144, R R 05514,
R R 00833, and by a grant from the Kroc Foundation.
James V. Dunne, M. D.: Assistant Professor of Medicine,
University of Ottawa, Ottawa General Hospital, Ottawa, Ontario,
Canada; Clarence J . Peters, M. D.: Virology Division, USAMRIID,
Fort Detrick, Maryland; Terry L. Moore, M. D.: Assistant Professor
of Medicine, St. Louis University, St. Louis, Missouri; John H .
Vaughan, M. D.: Head, Division of Clinical Immunology, Research
Institute of Scripps Clinic, La Jolla, California.
Address reprint requests to Dr. John H . Vaughan, Research
Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla
CA 92037.
Submitted for publication February 27, 1978; accepted
March 27, 1978.
Arthritis and Rheumatism, Vol. 21, No. 7 (September-October 1978)
of propranolol's membrane stabilizing effects and ability
to interfere with membrane receptor movement.
Many compounds have been used to modulate
humoral and cellular immune responses, sometimes as
biologic probes and sometimes as therapeutic agents.
Special interest has recently been shown in compounds
that affect the adenyl cyclase system (1-5), cellular microtubules and microfilaments (6-8), and membrane
stabilizing agents (9-12). We have recently reported a
hemolytic plaque-forming cell assay for the production
and release of a human autoantibody, rheumatoid factor (13). With this assay inhibitors of protein synthesis,
cycloheximide and puromycin, have been shown to interfere with production and release of rheumatoid factor
from peripheral blood leukocytes. Vinblastine, a disruptor of microtubules, also inhibits, as does D-propranolol, a membrane stabilizing compound (14).
In this report we have explored the effect of propranolol and some of its congeners on various lymphocyte functions that may be pertinent to its suppressive
effects on the lymphocyte membrane, inhibiting erythfactor. Adherence of red cells to lymphocyte membrane
receptors (rosette formation), patching and capping of
surface immunoglobulins (SIg), and the response of human lymphocytes to stimulation by plant mitogens have
been studied. Propranolol has demonstrated selective
effects on the lymphocyte membrane, inhibiting erythrocyte antibody (EA) rosettes, but not other rosettes.
SIg mobility in the membrane and responsiveness of the
lymphocytes to plant mitogens are both significantly
Lymphocytes. Lymphocytes from the peripheral blood
of normal healthy donors were purified by differential centrifugation on Ficoll-Hypaque (15), after which they were washed
once in balanced salt solution (BSS) and once in RPMI, then
suspended in RPMI 1640 with 1% fetal calf serum (FCS).
Adherent cells were removed by incubation at 37°C on plastic
dishes (Falcon) for one to 2 hours and decanting the nonadherent cells.
Macrophage Phagocytosis. Macrophage cell lines
BWJ-M and NZW-M were used. Twelve millimeter cover slips
were placed in the bottom of tissue culture wells (Falcon
3040). The mouse macrophages at a concentration of 5 X 105
in 0.3 ml RPMI 1640 with 10% FCS were added and incubated
for 4 hours at 37°C. Nonadherent cells were removed and the
adherent cells were incubated for 15 to 20 minutes in R P M I
1640 containing I % FCS and D- or L-propranolol or practolol
and 2.5 X lo-’ M, or
at concentrations of 1 X lo-‘, 5 X
in saline. Zymosan particles (Sigma) were diluted 1:25 in
RPMI 1640 with 1% FCS and the various concentrations of
drugs or saline were then added and incubated for 30 minutes
at 37°C. The cover slips were then washed and the cells fixed
with 1% glutaraldehyde, stained with Giems, and examined
microscopically. Cells containing three or more ingested particles were scored as positive.
EA Rosettes. Washed ox red blood cells (RBC) were
sensitized with a 1/50 dilution of a rabbit anti-ox RBC which
had a direct agglutinin titer of 1/32 and a Coombs’ titer of
1/512 (16). Lymphocytes a t 2.5 X 108/ml in RPMI 1640 and
1% FCS were mixed, with and without drug inhibitors, with an
equal volume of 1% sensitized ox RBC, spun at room temperature for 2 minutes at 500g and allowed to stand for 10 to 30
minutes. The buttons were then gently resuspended and the
number of rosettes in 300 to 400 live lymphocytes counted.
Only lymphocytes with three or more attached RBC were
counted as rosettes.
E Rosettes. Two-tenths milliliter of lymphocytes at 2.5
X 106/ml in RPMI 1640 and I% FCS were mixed, with and
without drug inhibition, with 0.2 ml of 0.2% SRBC and 0.1
ml of 9% Ficoll (17), spun a t 1000 rpm for 10 minutes, and
allowed to stabilize at room temperature for 60 minutes, then
resuspended and counted.
SIg Rosettes. For specific SIg rosettes, the spleen cells
of animals immunized intravenously 4 to 5 days previously
with 108 SRBC were suspended at 2.5 X 108/ml and mixed,
with and without drug inhibitors, with 0.2 ml of 0.2% SRBC,
spun at 1000 rpm for 10 minutes, and allowed to stand at room
temperature for 60 minutes. They were then resuspended and
the rosettes counted.
Patching and Capping of SIg. A n FITC-labeled F(ab’)2
fragment of a rabbit anti-human Fab was prepared as described (18). Lymphocytes were suspended in RPMI 1640 and
I% FCS at 0.5 X 10B/ml and were incubated at 4°C for 30
minutes with the FITC-labeled anti-Fab, with and without
inhibiting drugs present. The anti-Fab was washed out and the
cells were resuspended in RPMI 1640 and 1% FCS, again with
and without the drugs. The cells were then brought to room
temperature to allow patching and capping. The reaction was
stopped at 0, 5, 15, or 30 minutes by the addition of 0.5%
glutaraldehyde. The cells were observed in duplicate in a Carl
Zeiss fluorescence microscope with a UG-5 excitor filter, a no.
50 barrier filter, and an HBO-200W/4 mercury pressure lamp.
The readings were made blind and scored as follows:
r Rim-cells showing a complete rim of fluorescence
d Diffuse-very fine fluorescent stippling seen over all
the cell surface
p Patching-aggregates of fluorescence seen scattered
over the cell surface
c Capping-a rim or half-moon of fluorescence at one
cell pole only
DNA Synthesis. The plant mitogens used were phytohemagglutinin (PHA) reconstituted t o 5 ml in the diluent
provided (Difco), and pokeweed mitogen (PWM) reconstituted to 10 ml in the medium provided (Gibco). Lymphocytes
were suspended in sterile RPMI 1640 and 1070 heat inactivated
fetal calf serum at a density of 0.5 X 108 cells/ml. Twohundred microliter aliquots were added to individual wells of a
standard flatbottomed microtiter plate (Falcon). Drug inhibitors and plant mitogens were added in a total volume of 25 pl.
All conditions were examined in triplicate. The cells were
incubated for 72 hours at 37°C in a humidified atmosphere of
5% CO, in air. Five microcuries of Hs-thymidine (specific
activity 40 pCi/M) (New England Nuclear) were added and the
cultures were incubated for a further 18 hours. The cells were
harvested onto glass fiber filter paper using an automated cell
harvester (MASH), washed, precipitated with 6% trichloracetic acid, dried briefly, and transferred to counting vials
containing 10 ml instagel. The vials were counted in an automated scintillation counter. The stimulation index (SI) was
calculated as:
CPM responding cells
CPM control cells
Drugs Tested. D-propranolol, L-propranolol, and practolol were obtained from Ayerst Laboratories. Butoxamine
was obtained from Burroughs Wellcome, and vinblastine from
Eli Lilly. Congeners of propranolol were a-[(tert-butylamino)
methyl]-6-methyl-2 quindineethanol (MQE) and a-[(tert-butylamino) methyl]-6-phenanthridine-ethanol 2 HCI (PAE),
kindly provided by Dr. Robert Meyer (19) of Parke-Davis and
Viability Studies. The viability of cells after exposure
to the various drugs used was assessed by measurement of
lactic dehydrogenase (LDH) in the cell supernatants and by
estimating the number of cells that failed to exclude trypan
blue. Under the conditions used we did not detect LDH in
greater quantities in the supernatants of the cells exposed to
drugs than in those of the controls. There was greater than
80% viability by trypan blue staining, without differential effect between the test systems and the controls.
Both the D- and L- forms of propranolol at 10 X
A4 markedly inhibited the formation of EA rosettes
(Figure 1). Practolol and butoxamine, which have beta-1
and beta-2 adrenergic blocking activities but no membrane stabilizing properties, failed to inhibit. One congener of propranolol, PAE, which in previous studies
1052.5 1052.5 1052.5
Propranolol Propranolol
10 10 10
-o .-Er
"2 -Z E
e 3
Figure 1. Inhibition of EA rosette formation by D- and L-propranolol and the congener PAE. Practolol and
butoxamine. which have 0, and 0, adrenergic blocking activity but no membrane stabilizing activity.failed to inhibit.
The congener MQE. which had failed to inhibit rheumatoid factor production (ref 14). also failed to inhibit EA
M).The lines on the bars are f 2
rosettes. The numbers below each column are concentrations of drugs used ( X
(14) had shown an ability to inhibit the rheumatoid
factor plaque-forming cell assay, also inhibited EA
rosette formation. Another congener, MQE, which had
had little effect on the rheumatoid factor plaque-forming cell assay, failed to inhibit EA rosette formation. In
some experiments the lymphocytes were preincubated
for 30 minutes with the compounds to be tested and in
other experiments the compounds were added to the
lymphocytes together with the indicator red cells. This
variation in the experimental protocol did not change
the results found.
Experiments were conducted to determine how
rapidly the propranolol effect expresses itself. Lymphocytes and indicator red cells were mixed together at
room temperature in the presence and absence of propranolol and immediately spun into a button a t 500g.
The buttons were resuspended at 1,2,6, and 30 minutes
and the number of rosettes counted (Figure 2). The
inhibitory effect of propranolol was clearly evident at
one minute, which was the earliest sample that could be
I n separate experiments, washing the lymphocytes t o remove the drugs before addition of the red cells
restored their EA rosetting ability t o control values.
However, addition of propranolol to EA rosettes that
had been allowed to form and stabilize for 30 minutes
failed to disaggregate them.
To determine whether propranolol inhibits the
expression of other membrane receptors, its effects on Tcell rosette formation (E rosettes), rosette formation by
membrane-bound antibody (Slg) with antigen, and
mouse macrophage uptake of zymosan granules were
studied (Figure 3). In none of these systems did propranolol reduce the numbers of rosettes or ingested
particles from control values. Vinblastine, used as a
positive control in the mouse macrophage system,
markedly inhibited zymosan uptake. Vinblastine moderately inhibited E rosette formation, but did not inhibit
rosette formation by specific membrane-bound antibody.
The inhibitory effect of propranolol on EA rosette formation seems thus to be relatively specific, not
an effect on receptors generally. Since D-propranolol
worked as well as did L-propranolol, the membrane
stabilizing properties of propranolol rather than the
beta adrenergic blocking property seemed the likely
mechanism. Others (10) have noted that membrane stabilizing agents are capable of inhibiting patching and
capping of SIg of lymphocytes by F(ab'), anti-Fab. We
therefore studied D-propranolol similarly (see Materials
and Methods). The results are shown in Figure 4. In the
top panel the lymphocytes were mixed with the antiFab, kept at 4"C, and fixed with glutaraldehyde and
examined microscopically. In the presence of
tion of propranolol when incubated with the cells over
the 3 t o 4 day period of that experiment.
2 4 6 8 1 0
We have previously shown that D- and L-propranolol suppressed R F release by human lymphocytes
(14). In this report these observations have been extended to other lymphocyte functions. The effects of Dand L-propranolol, practolol, and butoxamine have
been compared. D-propranolol, which has membrane
stabilizing properties but essentially no 0-adrenergic
blocking activity, inhibits EA rosette formation, mitogen stimulation, and patching and capping of SIg. Practolol and butoxamine, which have PI and p2 blocking
properties, respectively, have no effect on EA rosette
formation, and butoxamine has also no effect on mitogen stimulation or patching and capping of SIg.
Figure 2. Kinetic s t d i e s on the inhibitory activity of D-propranolol on
EA rosette formation. The inhibitory effect was evident in the shortest
time interval at which the experiment could be done. The ordinate
represents the percent EA rosettes in the total lymphocyte population.
propranolol, the SIg remained diffusely distributed over
the cell surfaces. When the propranolol concentration
was reduced, more of the SIg appeared in patches and
caps. When the experiments were carried out with 5 to
I5 minute periods of incubation at 23°C prior to fixation, the principal difference noted was a considerably
increased patching and capping in the controls, which
the propranolol was partially but not completely able to
prevent. Butoxamine failed entirely to inhibit patching
and capping (Figure 5 ) .
Studies were carried out to determine whether
propranolol is capable of inhibiting the responses of
lymphocytes to plant mitogens. Phytohemagglutinin
and pokeweed mitogen were each used at three concentrations with propranolol at three concentrations. Dpropranolol reduced the responses of the lymphocytes
to both of these mitogens (Figure 6 ) . Butoxamine failed
to show a similar effect. At the end of the experiment,
trypan blue staining revealed 80% viability in the conMpropranolol. A t
trols and in 2.5 and 5 X
the viability and total cell recovery were reduced, indicating nonspecific cytotoxic effects of that concentra-
Mouse M*
5x10-5M lOxlO-5M
Figure 3. Failure of propranolol to inhibit E-rosette formation. zymosan
ingestion by mouse macrophages. and antigenically specific immunocyte
rosette/ormation (Slg rosettes). C = control; P = practolol; D = Dpropranolol; L = L-propranolol; V = uinblastine. In the bottom two
panels the ordinate represents percent of control. In the top panel the
ordinate represents percent of E rosettes in the total lymphocyte population. The lines on the bars are f 2 SEM.
77 1
5~10% 2.5~10.~1 ~10%
Figure 4. Inhibition of patching and capping of SIg by D-propmnolol. In
the top panel are the patterns of staining for surjbce immunoglobulins
found when peripheral blood lymphocytes were examined in the presence
and absence (Control) of propranolol; the temperature during the staining with anti-Fab and until the time of glutaraldehydefixation was kept
at 4°C (no time at 23°C). In the middle and bottom panels the cells were
allowed to stand for 5 and IS minutes at 23°C after staining and before
Jixation in glutaraldehyde. A marked inhibition of patching and capping
w s evident at 2.5 X
M propranolol and above. r = rim staining; d
= diffuse staining: p = patches; c = caps.
The inhibitory effect of D-propranolol on SIg
patching and capping confirms that propranolol affects
lymphocytes like other membrane stabilizing agents,
such as tranquilizers and local anesthetics (10). Patching
and capping depend upon the ability of the cell surface
receptors to move laterally within the membrane. It is
not known whether interference with this lateral movement by membrane stabilizing agents occurs through
“molecular packing” (20,21) of the surface membrane
because of the lipid solubility of the drugs in it, through
the disruption of microtubules and microfilaments that
are important to surface receptor movements (6-8), or
through drug charge effects that displace Ca++ and
other divalent metals from the membrane (22,23).
Whatever the mechanism, propranolol does interfere with membrane receptor movement, and this general
property may explain all the effects that have been seen
with the drug. For instance, propranolol may inhibit EA
rosette formation by preventing an organized movement
of membrane proteins to form effective Fc receptors
(6,24). It may explain the inhibition of the responses of
the lymphocyte to plant mitogens, if these responses
depend upon a signal to the cell from a cross-linking of
the surface receptors for the mitogens. The decreased
antibody production reported (14) for cells cultured in
propranolol probably is secondary to the inhibited mitogenic responses of the cells. Propranolol may inhibit
release of R F from RF-producing lymphocytes of the
peripheral blood, if the release depends upon the capping phenomenon and subsequent shedding, as appears
r dpc
D Propranolol
r d p c
Figure 5. Failure of the P-adrenergic agent. butoxamine. to inhibit patching and capping. The conditions were the
same as those of the top panel in Figure 4.
0101 or congeners developed from it may be made to
play a useful therapeutic role in rheumatoid arthritis or
other autoimmune disease remains to be seen. The
serum level of propranolol reached after a single oral
dose of 40 mg in patients is only about
A4 (31),
however, so extremely high doses would be needed. A
search among the congeners of propranolol for a better
therapeutic agent is probably merited.
We wish to acknowledge the excellent technical assistance of Ms Gail Sugimoto and grateful appreciation is due to
Ms Anna Milne who helped with the manuscript.
0 2.5 5.010.0
0 2.5 5.010.0
Propranolol [ X ~ O - ~ M ]
Figure 6. Inhibirion of stimulation of human peripheral blood lymphocyres b.v PHA and PWM in the presence of propranolol. Thedilurions of
niitogens used are indirared b.v the symbols in each panel and are the
dilutions of the srandard reconstiruted niitogen (see Materials and Merhods).
to be generally true for the release of surface Ig from B
cells (25).
The failure of propranolol to affect adversely E
rosette formation or antigen specific (SIg) rosette formation can probably be taken to mean that lateral movement of the receptors is not needed to develop the affinity of binding necessary for these rosetting procedures.
Failure of propranolol to inhibit phagocytosis by mouse
peritoneal macrophages is a confirmation that propran0101 in the doses used did not have significant short-term
cytotoxic effects of any generalized nature, a conclusion
also drawn for the lymphocytes by the cell viability
studies with trypan blue exclusion.
Drugs with membrane stabilizing activities are
capable of interfering with a number of cell-to-cell interactions, including platelet adhesiveness (26), cell fusion
(27), and leukocytic adherence to endothelium (28). Reduction of lysosomal enzyme release and superoxide
anion production by polymorphonuclear leukocytes
have also been described (29). Propranolol can be supposed to have some or all of these effects. Castor (30)
has noted a propranolol-induced decrease in production
of a connective tissue activating peptide (CTAP) by
inflammatory cells from joint fluids.
Whether these various effects on cells of propran-
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mitogen, effect, response, formation, rosette, differential, propranolol, plan, lymphocytes
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