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Alpha interferon accelerates lateral diffusion of Daudi cell surface differentiation antigens: Measurement by fluorescence redistribution after photobleaching
Vo1.157, No. 2,1988 December15,1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 808-8]5
ALPHA INTERFERON ACCELERATES LATERAL DIFFUSION OF DAUDI CELL SURFACE DIFFERENTIATION ANTIGENS: MEASUREMENT BY FL~OEESCENCE REDISTRIBUTION AFTER PHOTOBLEACHING Elisabeth Ballnt+, AdorJan Aszalos #, Philip M. Grimley Department o f P a t h o l o g y F. Edward H e b e r t M e d i c a l S c h o o l , USUHS; B e t h e s d a , Maryland
20814
Received October 24, 1988
SUMMARY Lateral diffusion coefficients (D) of two surface differentiation antigens (slgM and Bp35) were determined on interferon-sensitive (-IFs) or resistant (-IFr) Daudl cells by fluorescence photobleachlng, using monospeciflc FITC-anti-lgM or PE-anti~eu216 probes. For untreated Daudi -IYs, mea~n(D ~ were 5.8 and 5.3 (xl0- cm /set). These increased, toll and 7.9 x 10-'VcmZ/Rec (p 20.001) within 30 min after binding of recombinant IFN-a (80 to 800 U/10 v cells), but decreased by up to 4-fold after Con ~ Mean (D) of identical surface antigens on Daudi-IFr were 8.2 and 9.4 x I0- Vcm2/sec;and were not altered by IFN-a. Mean (D) of a lipid analog was up to 40-fold higher than for surface proteins and statistically identical in Daudi-IFs and Daudi-IFr. Rapid acceleration by IFN-a of surface protein lateral diffusion in Daudi-IYs obviously could facilitate antl-prollferative signal transduction; by contrast, a baseline increase of (D) in Daudi-IFr was evidently associated with their refractory state. ©igss AcademicPress,Inc.
Alpha interferons (IFN-a), which bind to specific high affinity receptors (I), represent a major group of the biologic response modifiers.
Their
pleiotroplc effects upon cell growth, enzyme activities, differentiation and immune functions (2-4) have been attributed to multiple response pathways
*The opinions or assertions expressed herein are private, unofficlal views of the authors, and do not necessarily represent views of the Uniformed Services University of the Health Sciences or of the Department of Defense. +Visiting Scientist from the Department of Biophysics, Attila Jozsef University, Szeged, Hungary. #Division of Drug Biology, Food and Drug Administration, Washington, D.C. Abbreviations: IFN-a, alpha interferon (generic); rlFN-a2, DNA-recombinant IFN-a, type 2; L-IFN-a, ultrapure IFN-a from human leukocytes; U, international anti-vlral units; Daudi-IFs, clone sensitive to growth inhibition by IFN-a; Daudi-IFr, subelone resistant to growth inhibition by IFN-a; FRAP, fluorescence redistribution after photobleachlng; (D), mean lateral diffusion coefficients; slgH, surface IgM; Bp35 (CD 20), 35,000 M.W. B-cell surface antigen; anti-Leu 16, mouse monoclonal antibody reactive with Bp35 (CD 20); PE, phycoerythrln B; FITC, fluorescein isothiocyanate; Con A, concanavalin A; NBD-PC, NBD-phosphatidylcholine; DMSO, dimethysulfoxide. 0006-291 X/88 $1.50 Copyright © 1988 by Academic Press, lnc. All rights of reproduction in any form reserved.
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(2,5), but specific signal transduction mechanisms remain unresolved (2,6-8). For prompt analyses of early plasma membrane changes in IFN-a-treated human lymphoblasts, we have utilized non-disruptive biophysical techniques. Previous work with potential sensing fluorescent dyes and flow cytometry demonstrated that Daudi-IPs cells, which are exquisitely susceptible to growth inhibition by IFN-a, responded to treatmentwith early changes in plasma membrane potassium ion flux (9,10).
This response did not occur in mutant
cells, Daudi-IFr, subcloned for resistance to growth inhibition (10).
We now
report results of fluorescence redistribution after photobleaching (FRAP). This technique quantitates lateral diffusion coefficients (D) of plasma membrane proteins such as receptors involved in signal transduction (11-15) or associated with ion channels (13-15).
MATERIALS AND METHODS Cell cultures: Daudi cells were grown in log-log suspensions (9,16) using EPMI 1640-25mMHepes with L-glutamine (GIBC0, Grand Island, NY), penicillin, streptomycin and 10~ Nu Serum (Collaborative Research, Cambridge, HA). The parental clone, Daudi-IFs, originated from a Burkitt's B-cell lymphoma (17), and is highly sensitive to growth inhibition by IFN-a (16,18). A subclone, Daudi-IFr, selected for spontaneous resistance (19), grew in medium wlth L-IFN-a (500 U/m1, 3 days/week). This resistance persisted for up to 12 mass doublings without L-IFN-a, and cells routinely were used after 3-5 doublings. They responded to induction of 2'-5'A synthetase (Grimley PM and Welsh D, unpublished results) as do other IFN-a-resistant subclones (~8-21). Reagents and fluorescent probes: Recombinant IFN-a2 (1.8xl0 U/mg protein) was a gift from Dr. T. Nagabushan (Schering CorPs, Bloomington, NJ). Ultrapure human leukocyte IFN-a (L-IFN-a, 2.7x10 U/mg protein) was purchased from Interferon Sciences, New Brunswick, NJ. Antl-viral ~nd antiproliferative activities were periodically verified with aliquots (4xl0 U/ml) stored a t -70 ° C (16). Mouse monoclonal anti-Leu-16, specific for Bp35 (CD 20) was obtained as a phycoerythrin-B (PE) conjugate (25 ~g i~munoglobulin/ml) from Benton Dickinson Monoclonals, Burlingame, CA. Column purified goat B-chain specific anti-human IgM, P(ab')~ (0.98 m 8 protein/ml), conjugated with FITC (absorbance ratio 1:1), and goat polyclonal anti-human IgM (1.0 mg protein[m1) were purchased from Tago (Burlingame, CA). Diluted reagents were centrifuged at Ixl0 x g (30 min) before use. Con A and valinomycin were obtained from Sigma Chemical Co., St. Louis, MO. Cyclosporin A was an FDA reference standard. NBD-PC solubilized in chloroform was purchased from Avanti Polar Lipids (Birmingham, AB), resolubilized in 95Z ethanol (4 mg/ml, cf. 22). A monoclonal mouse antibody to IFN-a w~s purchased from Boehringer Mannheim (Indianapolis, IN) and made up to 10 neutralizing units p~r ml. Fluorescent labeling and flow cytometrT: Aliquots of 2x[0 - cells were sedimented at 300 x g for 7 min, washed x[ in ice cold endotoxin-free Hank's BSS (Sigma Chemical Co., St. Louis, MO) with 3mM sodium azide (inhibits endocytosis and capping, cf. 21), then resuspended in 50 ~1 of the same buffer. Labelled antibody was added (5-20 ~1) for 30 min (anti-leu 16) or 50 min (anti-lgM) incubation (4°C). NBD-PC was diluted [:2000 (same buffer) and applied (4°C) for 30 min. Flow cytometry was performed with a Beckton-Dickinson FACScan System (15 mw argon ion laser, 488 nm excitation, and "C-30" software, Beckton Dickinson, Mountain View, CA). Cell cycle distribution was monitored by propidium iodide staining (23). Experimental treatments before FRAP: For most rapid analysis of 1FN-a effects, cells were labelled with fluorescent probe, incubated at 24°C in Hank's BSS (I00 ~i) and subjected to FRAP within 10 to 30 min. Some probe-labelled cells were identically treated with valinomycin (10 ~M in DM80,
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IZ v/v), cyclosporin A (I0 ~glml in DMSO, IZ v/v), or DMSO alone (1% v/v). In complementary analyses, cells were pre-lncubated with IFN-a in culture medium for 2 h at 37°C, then labelled with probes and re-equillbrated at 24@C. For experiments designed to alter lateral diffusion Con A (100 ~g/ml, 30 min, 24 ° C) or unlabelled polyclonal anti-lgM (200 ~glml, 50 min, 24°C) were introduced before or after probes or IFN-a. As a control for specificity, IFN-a was pre-mixed with a two-fold excess of neutralizing antibody. FEAP Technique: Cell suspensions (5 ~I) mounted in capillary microchambers remained 951 viable (trypan blue exclusion) for at least 4 h, and fresh aliquots were analyzed every 60 min at regulated ambient temperature (240C). Work was conducted at an ACA8 470 workstation (Meridian Instruments, Inc., 0kemos, MI) with an inverted microscope, computer controlled stage and 5W argon ion laser (Innova 90-5, Coherent, Inc., Palo Alto, CA) tuned to 488 nm. Power was adjusted between 160 to 400 mW. The collimated first order beam was reflected into an epi-illumlnation path through a standard dichrolc FITC filter cube (485/22 BP excitation filter, 510 DLP dichroic mirror, and 515 LP barrier filter), then attenuated 90~ by a neutral density filter. Fluorescent emissions were collected through a Zeiss Neofluor 63X objective (N.A. 1.25). An acousto-optic modulator (IntraAction Model 80A) provided for variable intensity laser scans or bleaching pulses. An integrated photomultiplier (Hamamatsu model 50-12) and data acquisition interface (80286 type) transformed fluorescence emissions from points of raster scans into 2-D pseudo-color images or transverse llne scans. For photobleaching, single cells with a uniform peripheral label were selected at random, and a laser pulse (48-120 ~J, Gaussian beam diameter
RESULTS As previously compared,
(cf. 18,19) cell cycle distributions of Daudi-IFs
and Daudi-IFr were similar under the log growth conditions essential for reproducible expression of IFN-a receptors (25). superimposable,
Scattergrams were
indicating comparable cell size distributions
(cf. 9) and
samples from both clones exhibited equivalent fluorescent intensities after saturation with FITC-anti-lgM (Fig. I). PE-anti-Leu 16.
Similar results were obtained with
Transverse laser scans of Daudi cells labelled with
FITC-anti-lgM or PE-anti-Leu-15 showed dual peaks of fluorescent intensity associated with the plasma membrane (Fig. 2) and minimal background fluorescence
(<2Z).
Reproducible asymptotic recoveries of fluorescence
( > 50Z
by 400-500 sec) followed single photobleachlng pulses (I0 msec) which reduced the original fluorescence by 40-70Z (Fig. 2).
Post-recovery surface integrity
was shown by repeated bleachlngs at the same spot (cf.26) and trypan blue exclusion.
Consistent means of (D) with each probe, modulation by Con A (see
below), and lack of labelling with non-speclfic PE or FITC-labelled antibodies, all indicated no significant binding of free fluorophore.
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750.
3000 d--A
2000
!
.
o
1000
.=
o
o
0
'
lbo
2~o
FluorescenceIntensity(channel#)
Q
20
40
do
L eo
lOO
ScanningDistance(1Pt= 0.25 Jam)
Fig. I Flow cytometry histograms co~paring Daudi cells incubated wlth ~-chain specific anti-lgM F(ab')? as described in Materlals and Methods: a) Daudi-IFs, antibody diluted 1:2.5 b) Daudi-lFs, dilution 1:10; c) Daudi-lFr, dilution 1:2.5 d) Daudi-IFr, dilution 1:10.
Fig. 2 Representative fluorescence intensity profiles from a Daudi-lFs cell probed with PE-anti-Leu 16, treated wlth T1~q-a2 and subjected to FRAP: a) Average of scans I-3 before photobleaching showing typical edge peaks (vertical lines) and waist diameter (ca 15~ m); b) Scan 4, I0 msec post-bleaching (58%), nots stable emissions from unbleached edge; c) Scan 28 of nearly completed recovery, 464 sec after photobleaching: the final recovery fraction (R=96%) was calc~ate~ by a normal mode analysis (24) and regression plot yielded (D)=7.14x10-'" cm /sec (correlation 0.96). (Decreased emissions from the opposite edge reflect dye redistribution and minimal secondary bleaching due to the repetitive low energy scans.)
Daudi-TFs:
Control (D) values obtained with FlTC-anti-lgMand
PE-anti-Leu-16
(Table I) matched ranges previously reported for surface proteins of B-cells (27), and also were similar without sodium azide pre-incubation.
As expected
(22), (D) values wlth the lipid probe, NBD-PC, were up 40-fold greater (Table 2).
l~l~-a2 treatment, for up to 30 mln after probe-labelling,
significantly increased the mean (D), both for slgH and Bp35 (Table I). similar effect on Bp35 occurred with L-TFN-a treatment (not shown).
A
The (D)
for sigh similarly increased when IFN-a incubation (2 h) preceded probe-labelllng
(Table I).
A log decrease of IF~-a concentration did not
alter results (not shown), but anti-TF~-a neutralization abolished the increase.
Changes in (D) after treatments with the hyperpolarlzing
ionophore,
valinomycin, or with cyclosporin A (cf.28), were similar to DMS0 alone (Table I).
In contrast, the (D) of both sIgMand Bp35 decreased nearly 4-fold when
treatments with Con A preceded or followed IFN-a2 (Table I), and mobile fractions also tended to decrease.
Pre-scan profiles (Fig. 2) confirmed the
stability of cell sizes during experimental treatments. Daudi-lFr cells:
Without treatment, (D) for sigh and Bp35 were consistently
higher than in untreated Daudi-lFs (Table I); however, no significant difference (Table 2).
(D) for NBD-PC showed
As ~n Daudi-IFs, Con A significantly
reduced (D) of the differentiation antigens (Table I); however, neither protein nor lipid diffusion responded to IFN-a2.
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TABLE i. Probe
SURFACE ANTIGEN DIFFUSION
Treatment #
FITC- a-lgM
PE- a-Leu 16
(D)
Daudi-IFs control IFN-a2 IFN-a2(*) Con A(*) Con A(*)/IFN-a2 IFN-a 2(*)/Con A Vallnomycin DMSO Cyclosporln A control IFN-a2 a-lgM(*)
N
p
R
80±20
5.8±2.3 11.0±2.8 10.1±1.4 2.5±0.6 3.0±0.4 3.1±0.7 6.7±0.3 6.4±0.5 6.6±1.4
44 29 15 15 I0 10 II 5 13
<0.001 " " " " N.S. " "
60±23 49±19 43±22 44±27 66±32 53±18 62±21
5.3±1.2 7.9±1.4 3.5±0.4
36 21 II
<0.001 "
54±24 62±27 51±19
66±23
Daudl-IFr FITC- a-lgM
control IFN-a2 Con A(*)
8.2±1.3 7.8¢1.0 3.1~0.5
22 15 I0
<0.001 60±29 N.S.@ 54±24 <0.001@ 65±15
PE- a-Leu 16
control IFN-a2
9.4±1.0 8.7±0.8
II II
<0.001 N.S.@
# = (*) (D) N p @ =
46±24 64±24
Rapid treatment after probe labelling unless indicated by (*) = Pre-incubation for 30 mln (Con A) or 2 h (IFN-%2) = Mean coefficient of lateral diffusion ±S.D., cm2/sec x I0 -I0 Number of cells measured by FRAP Probabillty compared to Daudi-IFs controls; N.S. = Not significant (p >0. I) Compared to Daudi-IYr control; R = Mobile fraction (recovery fraction)
DISCUSSION IFN-a2 consistently increased lateral diffusion of surface antigens on Daudi-IFs; whereas antigen diffusion on Daudi-IFr was already more rapid. Thus, initiation and discrimination of IFN-a actions may depend upon the fluid-molecular environment of surface membrane proteins.
Such changes
previously had been indicated by alterations in electron spin resonance (29), intramembrane partlcle distribution (30) and lipld ratios (31,32). Chromophore-antlbody pre-labelling is an inherent limitation of FRAP, since
TABLE 2.
LIPID PROBE DIFFUSION ¢
Cell Type
Treatment
Daudl IFs
Control IFN-a2
Daudl IFr
Control IFN-a2
(D~
N
R (%)
2.1±0.9 2.0±1.2
48 9
61±18 63±20
2.1±0.6 2.2±0.4
20 10
53±21 55±18
¢=NBD-PC, other abbreviations same as in Table I. °cm2/sec x 10 -8
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t h e p r o b e s t h e m s e l v e s c o n c e i v a b l y i n f l u e n c e membrane f l u i d i t y
(33,34): e.g.
b o t h sIgM and Bp35 can s e r v e a s transmembrane s i g n a l p o l y p e p t i d e s i n lymphocytes
(35-37).
Our experiments nevertheless showed t h a t diffusloual
changes remained consistent when IFN-a treatment p r e c e d e d probe-labelling (Table I), and preliminary tests with addltlonal monoclonal probes for B-cell differentiation antigens have yielded comparable results (unpublished data). PE has a relatively high molecular weight compared to FITC, but it proved to be a very stable fluorophore with high emission intensity (cf. 38, 39). While lateral diffusion can be sensitive to effective radii of receptor-probe complexes (II,12,14), differences in combined molecular weights of the fluorophores, antibodies and surface antigens did not appear to be major determinants of (D) values in the B-lymphoblasts
(cf. 27).
modulation (26,40,41), multlple receptor cross-llnking,
Anchorage
(II,33,42) or
increased membrane occupancy (43,44), all can retard lateral mobility of surface proteins; and one or more of these factors probably explains the effect of polyclonal anti-lgM (Table I).
Effects of Con A in decreasing (D),
before or after IFNa treatments, were of particular interest, since Con A acts on the cytoskeleton (40) and delays internalization of IFN-a (8).
Inhibition
of cell fusion and of capping of antlgen-antibody complexes by IFN-a (A5,46) has indicated that the cytoskeleton must be an ultimate target of action; however, in present experiments,
the effects of Con A and IFN-a2 on lateral
diffusion proved antagonistic at the early times when effector signals are generated.
IFN-a rapidly alters ion flux of Daudi-IFs cells, but not of
Daudi-IFr (9,10).
Present experiments indicate that increases in (D),
produced by IFN-a, are not solely related to ion flux: two other drugs that influence lymphocyte membrane potential (valinomycin or cyclosporin A) were not similarly effective.
Evidently, IFN-a relieves molecular diffusion
constraints by another mechanism,
such as propogated configurational changes
of transmembrane proteins (14,22,34,47).
Assuming that the anti-proliferative
signal involves a selective activation of ion channels (9,10), the prompt effect of IFN-a on molecular lateral motion in Daudi-IFs could thus be related to a facilitation of receptor-channel interactions and signal transduction (42).
The refractory status of Daudi-IFr remains to be explained, but a more
rapid baseline (D), as compared to Daudi-IFs, could be corollary to deficient receptor-cytoskeletal
connections such as those which have been detected in a
comparable Daudi subclone (7).
ACKNOWLEDGEMENTS The Daudi-IFr cells were a g i f t from Dr. Adl Kimchi, Welzmann Institute of Science, Eehovoth, Israel. T~:e authors are grateful to Hr. Scott Fine, Ms. Karen Fields, Ms. Bonnie Rupp and Hrs. Ellnore Dunphy for assistance. Supported by HAALT Grant GM 74-AQ from the Department of Defense.
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ALPHA INTERFERON ACCELERATES LATERAL DIFFUSION OF DAUDI CELL SURFACE DIFFERENTIATION ANTIGENS: MEASUREMENT BY FL~OEESCENCE REDISTRIBUTION AFTER PHOTOBLEACHING Elisabeth Ballnt+, AdorJan Aszalos #, Philip M. Grimley Department o f P a t h o l o g y F. Edward H e b e r t M e d i c a l S c h o o l , USUHS; B e t h e s d a , Maryland
20814
Received October 24, 1988
SUMMARY Lateral diffusion coefficients (D) of two surface differentiation antigens (slgM and Bp35) were determined on interferon-sensitive (-IFs) or resistant (-IFr) Daudl cells by fluorescence photobleachlng, using monospeciflc FITC-anti-lgM or PE-anti~eu216 probes. For untreated Daudi -IYs, mea~n(D ~ were 5.8 and 5.3 (xl0- cm /set). These increased, toll and 7.9 x 10-'VcmZ/Rec (p 20.001) within 30 min after binding of recombinant IFN-a (80 to 800 U/10 v cells), but decreased by up to 4-fold after Con ~ Mean (D) of identical surface antigens on Daudi-IFr were 8.2 and 9.4 x I0- Vcm2/sec;and were not altered by IFN-a. Mean (D) of a lipid analog was up to 40-fold higher than for surface proteins and statistically identical in Daudi-IFs and Daudi-IFr. Rapid acceleration by IFN-a of surface protein lateral diffusion in Daudi-IYs obviously could facilitate antl-prollferative signal transduction; by contrast, a baseline increase of (D) in Daudi-IFr was evidently associated with their refractory state. ©igss AcademicPress,Inc.
Alpha interferons (IFN-a), which bind to specific high affinity receptors (I), represent a major group of the biologic response modifiers.
Their
pleiotroplc effects upon cell growth, enzyme activities, differentiation and immune functions (2-4) have been attributed to multiple response pathways
*The opinions or assertions expressed herein are private, unofficlal views of the authors, and do not necessarily represent views of the Uniformed Services University of the Health Sciences or of the Department of Defense. +Visiting Scientist from the Department of Biophysics, Attila Jozsef University, Szeged, Hungary. #Division of Drug Biology, Food and Drug Administration, Washington, D.C. Abbreviations: IFN-a, alpha interferon (generic); rlFN-a2, DNA-recombinant IFN-a, type 2; L-IFN-a, ultrapure IFN-a from human leukocytes; U, international anti-vlral units; Daudi-IFs, clone sensitive to growth inhibition by IFN-a; Daudi-IFr, subelone resistant to growth inhibition by IFN-a; FRAP, fluorescence redistribution after photobleachlng; (D), mean lateral diffusion coefficients; slgH, surface IgM; Bp35 (CD 20), 35,000 M.W. B-cell surface antigen; anti-Leu 16, mouse monoclonal antibody reactive with Bp35 (CD 20); PE, phycoerythrln B; FITC, fluorescein isothiocyanate; Con A, concanavalin A; NBD-PC, NBD-phosphatidylcholine; DMSO, dimethysulfoxide. 0006-291 X/88 $1.50 Copyright © 1988 by Academic Press, lnc. All rights of reproduction in any form reserved.
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(2,5), but specific signal transduction mechanisms remain unresolved (2,6-8). For prompt analyses of early plasma membrane changes in IFN-a-treated human lymphoblasts, we have utilized non-disruptive biophysical techniques. Previous work with potential sensing fluorescent dyes and flow cytometry demonstrated that Daudi-IPs cells, which are exquisitely susceptible to growth inhibition by IFN-a, responded to treatmentwith early changes in plasma membrane potassium ion flux (9,10).
This response did not occur in mutant
cells, Daudi-IFr, subcloned for resistance to growth inhibition (10).
We now
report results of fluorescence redistribution after photobleaching (FRAP). This technique quantitates lateral diffusion coefficients (D) of plasma membrane proteins such as receptors involved in signal transduction (11-15) or associated with ion channels (13-15).
MATERIALS AND METHODS Cell cultures: Daudi cells were grown in log-log suspensions (9,16) using EPMI 1640-25mMHepes with L-glutamine (GIBC0, Grand Island, NY), penicillin, streptomycin and 10~ Nu Serum (Collaborative Research, Cambridge, HA). The parental clone, Daudi-IFs, originated from a Burkitt's B-cell lymphoma (17), and is highly sensitive to growth inhibition by IFN-a (16,18). A subclone, Daudi-IFr, selected for spontaneous resistance (19), grew in medium wlth L-IFN-a (500 U/m1, 3 days/week). This resistance persisted for up to 12 mass doublings without L-IFN-a, and cells routinely were used after 3-5 doublings. They responded to induction of 2'-5'A synthetase (Grimley PM and Welsh D, unpublished results) as do other IFN-a-resistant subclones (~8-21). Reagents and fluorescent probes: Recombinant IFN-a2 (1.8xl0 U/mg protein) was a gift from Dr. T. Nagabushan (Schering CorPs, Bloomington, NJ). Ultrapure human leukocyte IFN-a (L-IFN-a, 2.7x10 U/mg protein) was purchased from Interferon Sciences, New Brunswick, NJ. Antl-viral ~nd antiproliferative activities were periodically verified with aliquots (4xl0 U/ml) stored a t -70 ° C (16). Mouse monoclonal anti-Leu-16, specific for Bp35 (CD 20) was obtained as a phycoerythrin-B (PE) conjugate (25 ~g i~munoglobulin/ml) from Benton Dickinson Monoclonals, Burlingame, CA. Column purified goat B-chain specific anti-human IgM, P(ab')~ (0.98 m 8 protein/ml), conjugated with FITC (absorbance ratio 1:1), and goat polyclonal anti-human IgM (1.0 mg protein[m1) were purchased from Tago (Burlingame, CA). Diluted reagents were centrifuged at Ixl0 x g (30 min) before use. Con A and valinomycin were obtained from Sigma Chemical Co., St. Louis, MO. Cyclosporin A was an FDA reference standard. NBD-PC solubilized in chloroform was purchased from Avanti Polar Lipids (Birmingham, AB), resolubilized in 95Z ethanol (4 mg/ml, cf. 22). A monoclonal mouse antibody to IFN-a w~s purchased from Boehringer Mannheim (Indianapolis, IN) and made up to 10 neutralizing units p~r ml. Fluorescent labeling and flow cytometrT: Aliquots of 2x[0 - cells were sedimented at 300 x g for 7 min, washed x[ in ice cold endotoxin-free Hank's BSS (Sigma Chemical Co., St. Louis, MO) with 3mM sodium azide (inhibits endocytosis and capping, cf. 21), then resuspended in 50 ~1 of the same buffer. Labelled antibody was added (5-20 ~1) for 30 min (anti-leu 16) or 50 min (anti-lgM) incubation (4°C). NBD-PC was diluted [:2000 (same buffer) and applied (4°C) for 30 min. Flow cytometry was performed with a Beckton-Dickinson FACScan System (15 mw argon ion laser, 488 nm excitation, and "C-30" software, Beckton Dickinson, Mountain View, CA). Cell cycle distribution was monitored by propidium iodide staining (23). Experimental treatments before FRAP: For most rapid analysis of 1FN-a effects, cells were labelled with fluorescent probe, incubated at 24°C in Hank's BSS (I00 ~i) and subjected to FRAP within 10 to 30 min. Some probe-labelled cells were identically treated with valinomycin (10 ~M in DM80,
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IZ v/v), cyclosporin A (I0 ~glml in DMSO, IZ v/v), or DMSO alone (1% v/v). In complementary analyses, cells were pre-lncubated with IFN-a in culture medium for 2 h at 37°C, then labelled with probes and re-equillbrated at 24@C. For experiments designed to alter lateral diffusion Con A (100 ~g/ml, 30 min, 24 ° C) or unlabelled polyclonal anti-lgM (200 ~glml, 50 min, 24°C) were introduced before or after probes or IFN-a. As a control for specificity, IFN-a was pre-mixed with a two-fold excess of neutralizing antibody. FEAP Technique: Cell suspensions (5 ~I) mounted in capillary microchambers remained 951 viable (trypan blue exclusion) for at least 4 h, and fresh aliquots were analyzed every 60 min at regulated ambient temperature (240C). Work was conducted at an ACA8 470 workstation (Meridian Instruments, Inc., 0kemos, MI) with an inverted microscope, computer controlled stage and 5W argon ion laser (Innova 90-5, Coherent, Inc., Palo Alto, CA) tuned to 488 nm. Power was adjusted between 160 to 400 mW. The collimated first order beam was reflected into an epi-illumlnation path through a standard dichrolc FITC filter cube (485/22 BP excitation filter, 510 DLP dichroic mirror, and 515 LP barrier filter), then attenuated 90~ by a neutral density filter. Fluorescent emissions were collected through a Zeiss Neofluor 63X objective (N.A. 1.25). An acousto-optic modulator (IntraAction Model 80A) provided for variable intensity laser scans or bleaching pulses. An integrated photomultiplier (Hamamatsu model 50-12) and data acquisition interface (80286 type) transformed fluorescence emissions from points of raster scans into 2-D pseudo-color images or transverse llne scans. For photobleaching, single cells with a uniform peripheral label were selected at random, and a laser pulse (48-120 ~J, Gaussian beam diameter
RESULTS As previously compared,
(cf. 18,19) cell cycle distributions of Daudi-IFs
and Daudi-IFr were similar under the log growth conditions essential for reproducible expression of IFN-a receptors (25). superimposable,
Scattergrams were
indicating comparable cell size distributions
(cf. 9) and
samples from both clones exhibited equivalent fluorescent intensities after saturation with FITC-anti-lgM (Fig. I). PE-anti-Leu 16.
Similar results were obtained with
Transverse laser scans of Daudi cells labelled with
FITC-anti-lgM or PE-anti-Leu-15 showed dual peaks of fluorescent intensity associated with the plasma membrane (Fig. 2) and minimal background fluorescence
(<2Z).
Reproducible asymptotic recoveries of fluorescence
( > 50Z
by 400-500 sec) followed single photobleachlng pulses (I0 msec) which reduced the original fluorescence by 40-70Z (Fig. 2).
Post-recovery surface integrity
was shown by repeated bleachlngs at the same spot (cf.26) and trypan blue exclusion.
Consistent means of (D) with each probe, modulation by Con A (see
below), and lack of labelling with non-speclfic PE or FITC-labelled antibodies, all indicated no significant binding of free fluorophore.
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750.
3000 d--A
2000
!
.
o
1000
.=
o
o
0
'
lbo
2~o
FluorescenceIntensity(channel#)
Q
20
40
do
L eo
lOO
ScanningDistance(1Pt= 0.25 Jam)
Fig. I Flow cytometry histograms co~paring Daudi cells incubated wlth ~-chain specific anti-lgM F(ab')? as described in Materlals and Methods: a) Daudi-IFs, antibody diluted 1:2.5 b) Daudi-lFs, dilution 1:10; c) Daudi-lFr, dilution 1:2.5 d) Daudi-IFr, dilution 1:10.
Fig. 2 Representative fluorescence intensity profiles from a Daudi-lFs cell probed with PE-anti-Leu 16, treated wlth T1~q-a2 and subjected to FRAP: a) Average of scans I-3 before photobleaching showing typical edge peaks (vertical lines) and waist diameter (ca 15~ m); b) Scan 4, I0 msec post-bleaching (58%), nots stable emissions from unbleached edge; c) Scan 28 of nearly completed recovery, 464 sec after photobleaching: the final recovery fraction (R=96%) was calc~ate~ by a normal mode analysis (24) and regression plot yielded (D)=7.14x10-'" cm /sec (correlation 0.96). (Decreased emissions from the opposite edge reflect dye redistribution and minimal secondary bleaching due to the repetitive low energy scans.)
Daudi-TFs:
Control (D) values obtained with FlTC-anti-lgMand
PE-anti-Leu-16
(Table I) matched ranges previously reported for surface proteins of B-cells (27), and also were similar without sodium azide pre-incubation.
As expected
(22), (D) values wlth the lipid probe, NBD-PC, were up 40-fold greater (Table 2).
l~l~-a2 treatment, for up to 30 mln after probe-labelling,
significantly increased the mean (D), both for slgH and Bp35 (Table I). similar effect on Bp35 occurred with L-TFN-a treatment (not shown).
A
The (D)
for sigh similarly increased when IFN-a incubation (2 h) preceded probe-labelllng
(Table I).
A log decrease of IF~-a concentration did not
alter results (not shown), but anti-TF~-a neutralization abolished the increase.
Changes in (D) after treatments with the hyperpolarlzing
ionophore,
valinomycin, or with cyclosporin A (cf.28), were similar to DMS0 alone (Table I).
In contrast, the (D) of both sIgMand Bp35 decreased nearly 4-fold when
treatments with Con A preceded or followed IFN-a2 (Table I), and mobile fractions also tended to decrease.
Pre-scan profiles (Fig. 2) confirmed the
stability of cell sizes during experimental treatments. Daudi-lFr cells:
Without treatment, (D) for sigh and Bp35 were consistently
higher than in untreated Daudi-lFs (Table I); however, no significant difference (Table 2).
(D) for NBD-PC showed
As ~n Daudi-IFs, Con A significantly
reduced (D) of the differentiation antigens (Table I); however, neither protein nor lipid diffusion responded to IFN-a2.
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TABLE i. Probe
SURFACE ANTIGEN DIFFUSION
Treatment #
FITC- a-lgM
PE- a-Leu 16
(D)
Daudi-IFs control IFN-a2 IFN-a2(*) Con A(*) Con A(*)/IFN-a2 IFN-a 2(*)/Con A Vallnomycin DMSO Cyclosporln A control IFN-a2 a-lgM(*)
N
p
R
80±20
5.8±2.3 11.0±2.8 10.1±1.4 2.5±0.6 3.0±0.4 3.1±0.7 6.7±0.3 6.4±0.5 6.6±1.4
44 29 15 15 I0 10 II 5 13
<0.001 " " " " N.S. " "
60±23 49±19 43±22 44±27 66±32 53±18 62±21
5.3±1.2 7.9±1.4 3.5±0.4
36 21 II
<0.001 "
54±24 62±27 51±19
66±23
Daudl-IFr FITC- a-lgM
control IFN-a2 Con A(*)
8.2±1.3 7.8¢1.0 3.1~0.5
22 15 I0
<0.001 60±29 N.S.@ 54±24 <0.001@ 65±15
PE- a-Leu 16
control IFN-a2
9.4±1.0 8.7±0.8
II II
<0.001 N.S.@
# = (*) (D) N p @ =
46±24 64±24
Rapid treatment after probe labelling unless indicated by (*) = Pre-incubation for 30 mln (Con A) or 2 h (IFN-%2) = Mean coefficient of lateral diffusion ±S.D., cm2/sec x I0 -I0 Number of cells measured by FRAP Probabillty compared to Daudi-IFs controls; N.S. = Not significant (p >0. I) Compared to Daudi-IYr control; R = Mobile fraction (recovery fraction)
DISCUSSION IFN-a2 consistently increased lateral diffusion of surface antigens on Daudi-IFs; whereas antigen diffusion on Daudi-IFr was already more rapid. Thus, initiation and discrimination of IFN-a actions may depend upon the fluid-molecular environment of surface membrane proteins.
Such changes
previously had been indicated by alterations in electron spin resonance (29), intramembrane partlcle distribution (30) and lipld ratios (31,32). Chromophore-antlbody pre-labelling is an inherent limitation of FRAP, since
TABLE 2.
LIPID PROBE DIFFUSION ¢
Cell Type
Treatment
Daudl IFs
Control IFN-a2
Daudl IFr
Control IFN-a2
(D~
N
R (%)
2.1±0.9 2.0±1.2
48 9
61±18 63±20
2.1±0.6 2.2±0.4
20 10
53±21 55±18
¢=NBD-PC, other abbreviations same as in Table I. °cm2/sec x 10 -8
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t h e p r o b e s t h e m s e l v e s c o n c e i v a b l y i n f l u e n c e membrane f l u i d i t y
(33,34): e.g.
b o t h sIgM and Bp35 can s e r v e a s transmembrane s i g n a l p o l y p e p t i d e s i n lymphocytes
(35-37).
Our experiments nevertheless showed t h a t diffusloual
changes remained consistent when IFN-a treatment p r e c e d e d probe-labelling (Table I), and preliminary tests with addltlonal monoclonal probes for B-cell differentiation antigens have yielded comparable results (unpublished data). PE has a relatively high molecular weight compared to FITC, but it proved to be a very stable fluorophore with high emission intensity (cf. 38, 39). While lateral diffusion can be sensitive to effective radii of receptor-probe complexes (II,12,14), differences in combined molecular weights of the fluorophores, antibodies and surface antigens did not appear to be major determinants of (D) values in the B-lymphoblasts
(cf. 27).
modulation (26,40,41), multlple receptor cross-llnking,
Anchorage
(II,33,42) or
increased membrane occupancy (43,44), all can retard lateral mobility of surface proteins; and one or more of these factors probably explains the effect of polyclonal anti-lgM (Table I).
Effects of Con A in decreasing (D),
before or after IFNa treatments, were of particular interest, since Con A acts on the cytoskeleton (40) and delays internalization of IFN-a (8).
Inhibition
of cell fusion and of capping of antlgen-antibody complexes by IFN-a (A5,46) has indicated that the cytoskeleton must be an ultimate target of action; however, in present experiments,
the effects of Con A and IFN-a2 on lateral
diffusion proved antagonistic at the early times when effector signals are generated.
IFN-a rapidly alters ion flux of Daudi-IFs cells, but not of
Daudi-IFr (9,10).
Present experiments indicate that increases in (D),
produced by IFN-a, are not solely related to ion flux: two other drugs that influence lymphocyte membrane potential (valinomycin or cyclosporin A) were not similarly effective.
Evidently, IFN-a relieves molecular diffusion
constraints by another mechanism,
such as propogated configurational changes
of transmembrane proteins (14,22,34,47).
Assuming that the anti-proliferative
signal involves a selective activation of ion channels (9,10), the prompt effect of IFN-a on molecular lateral motion in Daudi-IFs could thus be related to a facilitation of receptor-channel interactions and signal transduction (42).
The refractory status of Daudi-IFr remains to be explained, but a more
rapid baseline (D), as compared to Daudi-IFs, could be corollary to deficient receptor-cytoskeletal
connections such as those which have been detected in a
comparable Daudi subclone (7).
ACKNOWLEDGEMENTS The Daudi-IFr cells were a g i f t from Dr. Adl Kimchi, Welzmann Institute of Science, Eehovoth, Israel. T~:e authors are grateful to Hr. Scott Fine, Ms. Karen Fields, Ms. Bonnie Rupp and Hrs. Ellnore Dunphy for assistance. Supported by HAALT Grant GM 74-AQ from the Department of Defense.
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