Clin. exp. Immunol. (1982) 49, 474-480.

E rosette dissociation: evidence for a role

of the cytoskeleton
D. BROHEE, B. KENNES & P. NEVE Laboratoire d'Immunologie Cellulaire, Clinique
Medicale Universitaire, Universit? Libre de Bruxelles, Centre Hospitalier 'Rene De Cooman',
Montigny-le-Tilleul, Belgium
(Acceptedfor publication 19 March 1982)

SUMMARY
When put into 370C incubation, the E rosettes dissociate spontaneously and the sheep
erythrocytes (SRBC) form caps at one pole of the lymphocytes. This process is associated
with changes in cell morphology such as uropod formation or membrane budding. The
disintegration of E rosettes and the capping of SRBC can be retarded by addition of
cytochalasin B plus colchicine, chlorpromazine or sodium azide. These findings suggest a
pivotal role of the cytoskeleton in the dissociation process of E rosettes. However, other
mechanisms of disintegration are to be considered since none of the drugs can prevent the
dissociation of E rosette entirely.

INTRODUCTION
Although the biological significance of this adherence is not yet fully understood, human T
lymphocytes form non-immune rosettes with various xenogeneic erythrocytes, especially with sheep
red blood cells (SRBC) (Hoffman & Kunkel, 1976). Emerging evidence suggests that the SRBC
receptors are membrane glycoproteins closely linked to surface antigenic determinants (Owen &
Fanger, 1974; Pyke, Rawlings & Gelfand, 1975; Galili, Klein & Schlesinger, 1977). The discrete
nature of the receptors is emphasized by experiments in which their partial isolation (Owen &
Fanger, 1974; Pyke et al., 1975) or their transfer to B lymphocytes (Jakobovits, Rosenberg &
Sharon, 1981) could be performed.
When put into 370C incubation, the E rosettes dissociate spontaneously and the SRBC are
drawn into caps over one pole of the lymphocytes (Yu, 1974; Cohnen & Brittinger, 1975; Cohnen,
1976; McMahon et al., 1976). Co-capping of the SRBC receptors can also be induced by some
antibodies (Owen & Fanger, 1974). Shedding, another feature of membrane proteins (Cone,
Marchalonis & Rolley, 1971; Black, 1980), also characterizes the SRBC receptors (Fernandez &
MacSween, 1977; Sdrmay, Istvain & Gergely, 1978). The fact that the receptors can be capped or
released into the medium lends some support to their dynamic state in a fluid membrane.
Recently using E rosette dissociation and SRBC-capping as easy means for studying membrane
dynamics, we have shown a restricted motility of the SRBC receptors of lymphocytes from elderly
humans (Brohee, Kennes & Neve, 1982), though no significant change in their affinity was observed
(Brohee et al., 1980). In a further step to determine the nature of this age-related alteration, the
question emerges whether the cytoskeleton is involved in E rosette dissociation and SRBC-capping.
Correspondence: Dr D. Brohee, Service de Medecine Interne, Centre Hospitalier 'Ren6 De Cooman',
C.G.T.R., rue de Gozee, 706, B-61 10 Montigny-le-Tilleul, Belgium.
0009-9104/82/0800-0474$02.00 © 1982 Blackwell Scientific Publications
474
 E-RFC and the cytoskeleton 475
In the present paper, we report the results of morphological and pharmacological investigations
supporting this contention.

MATERIALS AND METHODS

E rosetteformation. Peripheral blood lymphocytes (PBL) from healthy young volunteers (mean
27, range 16-35 years old) were drawn and prepared as previously reported (Kennes et al., 1981).
Batches of SRBC stored in Alsever's solution were purchased from Flow or Bioferm Laboratories.
Before use, the PBL and the SRBC were washed three times in Ca++ and Mg+ +-free Hank's
balanced salt solution (HBSS, Flow Lab.) and resuspended in HEPES-buffered RPMI 1640 medium
(Flow Lab.) respectively at 107/ml and 5 x 107/ml. E rosettes were formed by adding 100 MI of PBL
to 1 ml of SRBC, followed by centrifugation at 1000 rpm for 10 min and overnight incubation at
40C.
Kinetics of E rosette decay. After gentle manual resuspension, 200 yl aliquots were put into 370C
incubation for various periods of time. The investigated drugs were added just prior to incubation.
The dissociation process was stopped by adding equal volumes of 12% glutaraldehyde in PBS and
incubating the tubes in an ice bath for at least 30 min.
Drugs. Colchicine (CC) and cytochalasin B (CB) (stock solution in DMSO/saline) (Sigma Lab.)
were freshly diluted in HBSS and concomitantly used at 10-5 M (4 pg/ml) and 4-2 x 10-5 M (20
pg/ml) respectively (final concentrations). Chlorpromazine (CP) (Substantia Lab.) was prepared in
HBSS and used at i0-5 M final concentration. Different concentrations of PBS-dissolved sodium
azide (NaN3) were assessed (18 5-60 mM).
Lymphocyte morphology. Cells were examined in a Leitz Dialux 20 microscope equipped with
the Orthomat-W microphotography camera and photographs were taken with Kodak Tri-X or
Ilford PAN F 135 films.
E rosette enumeration and differential count. The PBL stained by diluted cresyl blue were counted
in a haemocytometer for at least 200 cells. A rosette (E-RFC) was defined as a lymphocyte with
three or more adherent SRBC. Cells in clumps were not considered. In the differential count, the
E-RFC were designated as morulae (M), horseshoes (H), caps (C) and indeterminates (I) as
previously described (Brohee et al., 1982, adapted from Yu, 1974) and shown in Fig. 1.
Statistics. The kinetics of E rosette dissociation and formation of the different types of rosettes
were summarized by the areas under the curves (integral calculus) with the number of E-RFC and
rosettes expressed in percentage of PBL. Each counting compared drug-treated and negative
control lymphocytes. The statistical significance of the difference was assessed using Wilcoxon's test
for paired data. In single paired experiments (with NaN3), the absolute counts of lymphocytes were
compared by the Chi-squared method.

b C

Fig. 1. Principal rosette formations: (a) morula with a complete ring of SRBC around the lymphocyte; (b)
horseshoe defined by one pole to half the circumference cleared from SRBC; (c) cap with SRBC gathered to less
than 180'. ( x 2000).
476 D. Brohee, B. Kennes & P. Neve
RESULTS
Changes in PBL morphology
As shown in Figs 2 & 3 capping was associated with changes in the lymphocyte morphology, mainly
heavy accumulation of SRBC at one pole of the cell giving the lymphocyte a turnip appearance with
its SRBC foliage and formation of a uropod with the capped SRBC at the top. Rarely, a small
budding of the cell membrane with thin villi anchoring SRBC was observed (not shown).
Drug effects
The synergistic action of colchicine and cytochalasin B significantly reduced the dissociation of E
rosettes (P = 0 01) (Fig. 4a). This stabilizing effect was already apparent at 10 min and lasted at least
until 20 min incubation. After this time, the dissociation curves became parallel. Moreover, in the
presence ofCC and CB, the morula-like formations were better preserved (P= 0-0 1), the proportion
of indeterminates increased (P = 0 01) whereas the capping of SRBC was inhibited (P = 0 01) (Fig.
4b).
As depicted in Fig. 5, chlorpromazine also inhibited E rosette dissociation though a little later.
No significant difference existed between both curves, but at 30 min, the number of E-RFC was
significantly greater after adding CP (39-3 + 3 7, mean + s.e.m., against 29 7 + 4-4, P= 0 01).
Chlorpromazine retarded the dissociation of morulae (P = 002), the formation of caps
(P = 0-01) and tended to enhance indeterminate rosettes (not significant) (Fig. 5b). Stabilization of
all rosette types was observed between 15 and 30 min. An obvious rounding of PBL and SRBC
occurred with CP (not shown).

Fig. 2. Lymphocyte morphological changes associated with SRBC capping. Heavy accumulation of SRBC at
one pole (turnip appearance). ( .x
1100).

Fig. 3. Lymphocyte morphological changes associated with SRBC capping. Uropod formation with SRBC at
the top (a and b x 900; c x 2000).
 E-RFC and the cytoskeleton 477
60 (a) 50 (b)
30=
X morulo
c 240
0
IL10-neemnt

Time (min)
Fig. 4. Effect of colchicine (4 yg/ml) plus cytochalasin B (20 yg/ml). (a): slower rate of dissociation of the E
rosettes. (b): stabilization of morulae, inhibition of capping, increased formation of indeterminates (0-0
treated cells, control cells; the vertical bars represent s.e.m.).

80 - (a ) 30

_ 10- morulo
60L0 I 10 L
080 0()30-20 45 0 10 2
(
Fig. 4. Effect of coihicprmaine 4 4gmi
60~~~~~~~ plus cytochlatestainizBt(20 og/i) (a) sloweter. praesofdisocation of thule En
TimeCL(min.
apn cells, *-* control cells; the vertical bars represent s.e.m.).
iniiino a0treated
10
no te

o 0)

0 15 30 2 0 5 3

10m indtemiat

Fig. 5. Effect of chlorpromazine (3-4 pg/mi). (a) late stabilization of E rosettes. (b) preservation of morulae and
inhibition of capping (0-01 treated cells, 0-0 control cells; the vertical bars represent s.e.m.).

Sodium azide also inhibited the process of E rosette dissociation, but required relatively high
concentrations (Table 1), and capping of the SRBC was reduced. This drug effect was evident after
15 min incubation and persisted, though at a lower level, after 30 min (not shown). Cell rounding
occurred at the highest NaN3 concentrations.

DISCUSSION
One of the most important features of the cellular membrane is that proteins can move laterally
within the plane of the membrane or be released into the medium (Lackie, 1980; Loor, 1979; Black,
1980). Furthermore, when cells are incubated with multivalent ligands, the randomly distributed
receptors are cross-linked into discrete patches that are subsequently dragged over one pole of the
cell and form caps. This latter process is energy dependent and involves the contractile proteins of
the cell (Nicolson & Poste, 1976; Loor, 1979; Lackie, 1980). In lymphocytes, capping occurs with
478 D. Brohee, B. Kennes & P. Neve
Table 1. Effect of increasing concentrations of sodium azide on E rosette dissociation and SRBC-capping after
incubation at 370C for 15 min (single paired experiments)

% Inhibition
NaN3 (mM) of dissociation* (P)t % Inhibition of capping: (P)§

18 5 15.5% (n.s.) 6777% (n.s.)

308 -1-4%(n.s.) 73 1%(<0.02)
46-2 30 1% (<0 02) 99-6% (<0 001)
60-0 55.3% (<0001) 6622% (<001)
* Calculated as
F100- 100 X percentage E-RFC (15', NaN3) -percentage E-RFC (0')
L percentage E-RFC (15', control)-percentage E-RFC (0')]
E-RFC expressed in percentage of PBL.
t Chi-squared (E-RFC/not E-RFC, NaN3/control).
100-1 OOX percentage caps ( 15', NaN3) -percentage caps (0') 1
L percentage caps (15', control) -percentage caps (O') caps expressed in percentage of E-RFC
§ Chi-squared (M/H/C/I, NaN3/control).

various proteins, for example, membrane immunoglobulins, Fc receptors, histocompatibility

antigens, some T cell specific xenoantigens and various glycoproteins bound by concanavalin A
(Kourilsky et al., 1972; De Petris, 1975; Braun et al., 1978a).
In the present study, we demonstrate that SRBC behave like multivalent cross-linking agents
and induce their own capping by lymphocytes. This process is associated with changes in cell
morphology (Figs 2 & 3) and is inhibitable by colchicine plus cytochalasin B (Fig. 4), synergistic
agents in their antagonistic effect on microfilament functions (De Petris, 1974). Chlorpromazine,
which interacts with membrane phospholipids, modulates Ca+ + ions and phosphodiesterase and
finally disconnects both microtubules and microfilaments from the cell membrane (Ryan, Unanue
& Karnovsky, 1974; Levin & Weiss, 1976; Schliwa et al., 1981), also inhibits E-RFC disintegration
and SRBC capping (Fig. 5). Finally sodium azide, a poison of the mitochondrial ATP synthesis, is
able to block capping mechanisms (Table 1). These results provide indirect evidence for the pivotal
role of the cytoskeleton in the dissociation process of E rosettes.
Dissociation of E rosettes and capping of SRBC occur very rapidly. Indeed the time elapsed for
resuspension of rosettes, distribution of aliquots and fixation by glutaraldehyde is sufficient for
advanced disintegration, since the number of E-RFC and the proportion of complete morula-like
rosettes are significantly lower than in counts performed immediately after resuspension (Brohee et
al., 1982). Cap formation seems to reach a plateau between 15 and 30 min at 37°C (Figs 4 & 5). The
proportion of horseshoes never changes significantly, indicating the very transient nature of this
formation.
As far as CC and CB are concerned in specific inhibition of microtubule and microfilament
functions, two dissociation phases should be differentiated: the first one occurring relatively early
and inhibitable by these drugs, and a second one, mostly evident after 20 min, going on despite the
presence of CC and CB.
In contrast to the CC-CB association, chlorpromazine exhibits a slower stabilizing effect. We
suppose that its action occurs at a later step of the capping process or that CP induces other effects
such as increasing membrane fluidity with subsequent enhanced shedding or faster type II lateral
diffusion and capping (Braun et al., 1978b) of the SRBC receptors. These processes are likely to exist
since none of the tested inhibitors could abolish the dissociation of E rosettes entirely. Indeed, the
increase in the proportion of indeterminate rosettes observed with CC-CB, CP and NaN3, do
suggest a random release of the receptors.
In comparison to other studies, the concentrations of NaN3 required to inhibit SRBC capping
 E-RFC and the cytoskeleton 479
and rosette dissociation may appear somewhat excessive. However cap formation is closely linked
to cellular ATP levels and in human lymphocytes, mitochondrial inhibitors are relatively ineffective
in the presence of glucose (Pozzan et al., 1980) (our experiments were performed in RPMI 1640
nutritive medium).
It seems relevant to our study that E rosette formation is optimal at 40C, a temperature that
blocks cellular active metabolism and disrupts microtubules (Nicolson & Poste, 1976). Recent
experiments supporting the similarity of action between zinc and CB on the cytoskeletal function
(Rao & Schwartz, 1980), shed a new light on a previous report in which zinc was shown to stabilize E
rosettes and to slacken SRBC capping (McMahon et al., 1976).
Since SRBC capping is closely related to the cytoskeleton, some transmembrane message seems
likely. Indeed several studies lend some support to this contention. Recently, it has been shown that
lymphocytes involved in E rosette formation were activated (Larsson, Andersson & Coutinho,
1978; Silva et al., 1981).
As a corollary, activated cells form stable rosettes at 370C (Galili & Schlesinger, 1976; Pandolfi
et al., 1980). This indicates some locking of the membrane, a phenomenon akin to that described in
mouse lymphocytes tolerant to trinitrophenyl determinants (Ashman & Naor, 1979) or incubated
with Con A (De Petris, 1975).
Although other membrane functions or properties may also intervene, E rosette dissociation
and SRBC capping provide easy tools for studying the microfilament-microtubular system in
human T lymphocytes and for getting some insight on the cytoplasm-membrane interactions.

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