0022-1767/90/1445-1640$02.00/0
THEJOURNAL
OF IMMUNOLOGY
Copyright 0 1990 by The American Association of immunologists
Vol. 144. 1640-1645. No. 5. March 1. 1990
Printed In U.S.A.
MONOVALENT LIGANDSOF COMPLEMENT RECEPTOR 2 INHIBIT WHEREAS
POLYVALENTLIGANDS ENHANCE ANTI-@-INDUCED HUMAN B CELL
INTRACYTOPLASMIC FREE CALCIUMCONCENTRATION'
GEORGE c. TSOKOS,2*tJOHND.
LAMBRIS,* FRED D. FINKELMAN,'
EVANGELOS D. ANASTASSIOU,+ AND CARL H. JUNE'
From the *Departmentof Medicine. Uniformed Services University of the Health Sciences, Bethesda,
MD 20814: 'Kidney
Diseases Section.National Institute of Diabetes. Digestive and Kidney Diseases,
National Institutes of Health, Bethesda,MD
20892:'Basel Institutefor Immunology, 4005 Basel, Switzerland, 'Naval
Medical Research Institute. Bethesda,MD 20814
We have performed experiments
to investigate the CR2, including the murinemAb HB5, have been produced
role of ligands for complement receptor 2 (CR2) in (4, 8).
human B cell activation.Flow microfluorimetry was CR2 has been considered to play a role in B cell differused to assess changes in free intracytoplasmic
cal- entiation and proliferation. Polyclonal and monoclonal
ciumconcentration[Ca"'] in indo-loaded B cells, antibodies to CR2 (11-13) a s well as particle bound C3d
immediately after exposure to anti-p antibody and (14) have been shown to enhance B cell responses in
to monovalent or polyvalent CR2 ligands. A s mon- different systems. More recently, HB5 has been found to
ovalent ligandswe used theC3d fragment andsyn- act synergistically with p-chain-specific antibody to inthetic C3 peptides(peptidesP14,residues1201crease B cell intracytoplasmic free calcium ([Ca2+]i) (15).
1214, and P28, residues 1187-1214).A s polyvalent
Monomeric fluid phase C3d, in contrast, hasbeen found
ligands we usedi) an intact monoclonal mouse antiCR2 antibody (HB5) and its F(ab')" fragment, ii)
tet- to inhibit murine B cell proliferation (14). P14 and P28
ravalent P13 ((residues 1202-1214) 4-template),
and peptides inhibit the maturationof murine B cell progeniii) P28 conjugated to BSA (molar ratio 5/1).Anti- itors (16) while they support the growth of CR2-binding
CR2 antibody HB5, tetravalent P13, and P28 conju- EBV lymphoblastoid B cell lines (10 ) .
gated to BSA, enhanced the ability of F(ab')" frag- Cross-linking of B cell surface Ig induces hydrolysis of
ments of the IgG fraction of goat anti-human p an- phosphatidylinositol 4,5-bisphosphate by phospolipase C
In contrast, to inositol trisphosphate anddiacylglycerol. Inositol tristibody to increase humanB cell [Ca"+]i.
the monomeric CR2 ligands C3d and P28 inhibited phosphate causes the release of intracellular calcium
the anti-p-induced increase in human
B cell [Ca"+]i. from endoplasmic reticulum (17-21). [Ca"+]ican be acMultivalent P13, P28, and the HB5, by themselves, curatelymeasured in isolated B cells using the Ca2+did not affect B cell (Ca"+]i. These experiments sugdependent indicator indo- 1 (2 1, 22).
gest that the valence of the CR2 ligands is crucial
In this study we used the anti-p-induced increase in
for the nature (synergistic vs antagonistic) of the [Ca"']i, a n early event in B cell activation, to study the
message transmitted through the
CR2.
role of CR2 in B cell function. We found that CR2-mediated modulation of B cell activation is ligand-valencydependent, in that monomeric ligands inhibit the anti-pCR23 is a 140-kDa glycoprotein (1-3) that specifically
induced increase in [Ca2']i whereas polyvalent CR2 libinds iC3b, C3dg. and C3d fragments of C 3 (4, 5)and the
gands enhance theability of anti-p to increase [Ca"+]i.
EBV envelope protein gp350/220 (6,7). CR2 is expressed
primarily by B cells, although it has been found on other
MATERIALS AND METHODS
cell types (5,8).
CR2 binds a site on C 3 that is composed of residues
Cells. Peripheral blood MNC were obtained from heparinized blood
1205- 12
14 of the C 3 sequence (9).Synthetic C3peptides by Ficoll-Hypaque density centrifugation. Human spleen cells were
through the Tissue Procurement Service of the Clinical
P14 (residues 1201- 12 14) and P28
(residues 1 18712 14) obtained
Center of the National Institutes of Health.
bind to CR2 (9, 10) and canbe used a s ligands in funcReagents. Affinity-purified F(ab')2 fragmentsof the IgG fraction
tional assays.A number of murine mAb that bind human of goat anti-human P (purchased from Jackson ImmunoResearch
Laboratories, West Grove, PA) were used to stimulate B cells. PElabeled monoclonal mouse IgGlk anti-human CD20 antibody was
Received for publicationJuly 5. 1989.
purchased from Becton Dickinson (Mountain View, CA). PE-labeled
Accepted for publication November 29. 1989.
Leu-4, Leu-M3, Leu-7, and Leu-1 1 (Becton Dickinson) were used in
The costs of publication of this article were defrayed in part by the
some experiments. HB5 mouse lgG2ak anti-CR2 was obtained from
payment of page charges. This article must therefore be hereby marked the American Type Culture Collection, Rockville, MD. F(ab'), fragadvertisement in accordance with 18 U.S.C. Section 1734 solely to indiments of HB5 were prepared by pepsin digestion and separated from
cate this fact.
undigested IgG2ak molecules by absorption with protein A SephaThis work was supported in part by Uniformed Services Universityof
rose (Pharmacia, Piscataway. NJ). Purity was demonstratedby SDSthe Health Sciences Grant C083EU. The Basel lnstitute of Immunology PAGE analysis. F(ab'), fragments of the IgG fraction of goat antiwas foundedand is entirely supported by F. Hoffmann-LaRoche Ltd.Co..
murine IgG antibody were purchased from Cappel-Cooper BiomediBasel. Switzerland.
'Address correspondenceand reprint requests to Dr. George C. Tsokos. cals (Malvern. PA). F(ab'), fragments of goat anti-human-p and/or
HB5 antibodies were bound onto cyanogen bromide-activated SephNIH. Bldg. IO, Rm. 3N112. Bethsesda. MD 20892.
Abbreviations used in this paper: CR. complement receptor: [Ca2+]i. arose (Pharmacia)according to theprovided instructions.
C3 was prepared from human plasma and C3d was prepared by
intracytoplasmic free calcium concentration; MNC, mononuclear cells:
digestion as previously described (23). C3-derived CR2-binding pepPE. phycoerythrin.
~
~
~
~
~
~~
1640
CR2
AND
LIGANDS
tides (P14 and
P28) were prepareda s previously detailed (9, 10).Both
bind to CR2 but P28 bindswith a higher affinity thandoes P14 (9).
The P13-template was synthesized
according to the principle of
Mutter et al. (24)as previously detailed (10).In brief, the amino acid
template was synthesized first on a resin by using butyloxylcarbonyl-Lys(F-moc)-OHfor four cycles. Four P13 peptides were synthesized on the template-resin afterdeprotection of the F-moc protected sites.Thefinal
product was cleaved from theresinand
purified by gel filtration. It was calculated that the majority (>go%)
of the CR2 were occupied when P28 was used at concentrations
greater than 60 pg/ml in cell suspensions of 1 X 106/ml (10).P28. to
which a cysteine residue was added to the amino-terminus, was
conjugated to BSA as described by Lerner et al. (25).The P28:BSA
molar ratio was calculated to be 5: 1.
Intracellular lonlzed calclum a s s a y . [Ca2+]iin single cells was
measured as described (26, 27). Briefly, cells were loaded with the
acetoxymethyl ester of indo-1 (Molecular Probes, Eugene,OR). Initial
concentrations of 3 pM of this compound, resulted inan intracellular
indo-1 concentration of 20 to 50 p M . After the loading procedurethe
cells were washed, placed in fresh medium at 2.5 x 106/ml and
stored in the dark at room temperature until analysis. Immediately
before each assay, indo-1-loaded cells were diluted
to 1 X 106/ml
with medium (HBSS, containing 1 mM CaCl,, 1 mMMgCl,, 20 mM
HEPES, and 10% FCS). equilibrated a t 37°C and analyzed by flow
cytometry at 400 to 500 cells/s. For each cell analyzed the ratio of
vio1et:blue fluorescence was digitally calculated in real time. The
[Ca2']i (nM) was calculated from the indo-1 violet to blue ratio after
proper calibrations were performed as previously detailed (27). Before loading with indo-l thecells were stained with PE-labeled antiCD20 or with a mixture of PE-labeled anti-Leu-4, Leu-M3, Leu-7,
and Leu-11 by incubating for 30 min a t 4°C. This permitted the
identification (gating] of B cells by positive or negative selection,
respectively. Preliminary experimentsdemonstratedthat
prior
staining of cells with anti-CD20 antibody had no effect on the anti#-induced increase in [Ca2')i.
Proltferatlon a s s a y s . B cells (70 to 80% CDZO'] were enriched
from peripheral blood or spleen by depletion of monocytes through
plastic adherence and T cells through sheep erythrocyte rosetting
and werecultured in 96-wellround-bottommicrotiterplates
at
50,000/well in medium RPMI 1640 containing 10%FCS. Cells were
harvested 72 h after the initiation of the culture and were pulsed
with 0.5 pCi/well of 3H-thymidine for 18 h before harvesting.
RESULTS
Cross-linking of IgM with F(ab'), fragments
of goat
anti-IgM antibody leads to a dose-dependent increase in
[Ca2']i. In a typical experiment (Fig. 1) peripheral blood
MNC were stained first with PE-labeled anti-CD2O and
then loaded with indo-1 . They were equilibrateda t 37°C
before use and exposed to graded doses of F(ab')z goat
Boot
1641
B CELL ACTIVATION
anti-human p . (Caz']i was observed to increase instantly
and decline and plateau a few minutes later; similar
responses were obtained from
MNC from 14 different
normal individuals and four different spleen
B cell preparations that were studied during the course
of these
experiments. Peripheral cells were stained first with a
mixture of PE-labeled anti-leu 4, leu-M3, leu-7 and leu1 1 . [negative selection) or with
anti-CD2O (positive selection) before loading with indo-1 and analysis. Positively
or negatively gated cells responded similarly to anti-p.
To investigate the effect of monovalent CR2 ligands on
the anti-p-induced increaseof [Ca2+]iwe first exposed the
cells to graded concentrations
of C3d, the natural ligand
of CR2, and then added anti-1 antibody. A s shown in
Figure 2, preincubation of cells with C3d for 5 min a t
37°C inhibits the ability
of 10 pg/ml of anti-1 antibody to
increase [Ca2']i. In four more experiments, which were
performed with either peripheral MNC (twice) or spleen
cells (twice), C3d (60 @/ml) inhibited the anti-p-induced
[Ca"]i response by 40 to 62%. C3d inhibition of the B cell
[Ca"+]iresponse was greatest when the anti-p antibody
was used at 10 pg/ml and less athigher and lower anti-p
concentrations (Table I). Preincubation of the cells with
C3d for longer periods of time (up to 20 min), inhibited
the anti-p response to the same extent.
Addition of C3d,
simultaneously with the anti-p antibody, had a less reproducible inhibitory effect. Additionof C3d alone at the
time of the initiation of the analysis had no effect on B
1
700
- 600
:
- 500-
.-c0
2
400 -
aY
0
6
V
E
300 -
$
- 200 -
I
I
10
20
4
I
I
30
40
50
Time
Figure 2. Inhibition of the anti-#-induced increase in B cell [Cazc]iby
C3d. Peripheral MNC were stained with PE-anti-CD20. loaded with indo1 and equilibrated at 37°C before analysis. They were treated with 10 pp/
ml 1,-(
30 p d m l [ . . . . . .), or 60 puml 1. -. - , - . -) of C3d. or were left
untreated 1- - - - -) before the addition of 10 #@nl of Flab'),-goat anti-@.
TABLE 1
Inhibition of anti-p induced increase in [Caz+]iofB cell-enriched
DeriPheral blood MNC bu the CR2 liaand P 2 8
Concentration of F(ab'), Anti-p Antibody
[pg/ml)
~
3
Time
Figure 1 . Peripheral MNC were first stained with
PE-anti-CDZO,
loaded with indo-1 and equilibrated to 37°C before analysis. Flab'),-goatdifferent
anti-human p was added to cells at the indicated time[arrow) at
30 &/ml. The
concentrations: (.-.-.). 3 pglml; (. . . .). 10 pgjml: (----I.
interval 0-100 on the abscissa is equivalent to 6 min. The concentration
of Ca" was calculated as described in Materials and Methods and is
expressed as n M .
No ligand
10
30
309"
455
570
230 (44)
260 (60)
455 (26)
280 (16)
270 (56)
470 (23)
Maximum recorded channel [Caa+]i[nM). B cell-enriched peripheral
blood MNC were incubated with or without 100 pg/ml of P28 for 5 min at
37°C before stimulation with the indicated concentrations of anti-@ antibody.Numbersin
parentheses indicate the % of inhibition that was
calculated after subtracting the baseline ICa2+]i( 130 nM).
60 pglml C3d
100 pgjml P28
1642
CR2 LIGANDS AND B CELL ACTIVATION
cell [Ca"+]i(Fig. 3).
The above experiment was repeated, substituting for
C3d the peptides P 14 (residues 1201- 1214 of C3) or P28
(residues 1 187-1214 of C3). Preincubation of cells with
P14 had little effect on the anti-p-induced increase in
[Ca"']i (Fig. 4). Prolongation of this preincubation timeup
to 30 min still had little effect. Similarly, P14 failed to
inhibit the anti-p-induced human spleen B cell[Ca2']i
response (data not shown). In contrast, preincubation of
cells for 5 min at 37°C with graded doses of P28, which
is bound by CR2 with higher affinitythan P14 (9).inhibited the anti-p-induced increases of [Ca"+]iin a dose00 pg/ml) inhibited the
dependent manner (Fig. 5). P28 (1
anti-p-induced [Ca2+]i responsein six more experiments
"0
10420
40
Time
30
50
60
FLgure5. The CR2-binding peptide P28 inhibits the anti-winduced
increase in[Ca"]i. Peripheral MNC were treated as in the legend to Figure
2. After equilibrationa t 37°C they were treated with
(- - - - -), 50 pm/ml:
or (-. -. -. - .). 100 pgmlof P28. or (-),
no P28, for5 min before the
addition of 10 pg/rnl of F(ab'),-goat anti-p ( a r r o w ] .
I
L
i
10 "0
420
30
40
50
Time
FLgure 3. Monovalent or polyvalent CR2 ligands have no effect on
the
B cell [Ca'+]i when present in the absenceof anti-p antibody. Peripheral
MNC were stained with PE-anti-CD2O. loaded with indo-1 and equilibrated at37°C before analysis. At the position of the arrowwe added: 30
pg/ml anti-p (-----). 100 pglrnl P28 (. . . . . .). 60 pglml C3d (-.-.-.), 1 p g l
ml HB5 I- - - -), or 50 pg/ml of (P13)4-ternplate(-).
700
600
using peripheral MNC and 2 more using spleen cells; the
percentage of inhibitionranged from 45 to 65%. A s
shown in Table I, P28inhibited optimally the [Ca2']i
response, when the anti-p antibody was used at 10 pg/
ml and to a lesser extent when higher or lower concentrations were used (Table I). Addition of either P14 or P28
at the initiation of the analysis, in the absence
of anti-p
antibody, had no effect on [Ca"+]i(Fig. 3).These experiments show clearly that monovalent CR2 ligands inhibit
a n early, anti-p-induced, B cell activation event, the increase of [Ca"]i.
Because polyvalent CR2 ligands have been reported to
enhance B cell responses we next investigated the effect
of exposure of human peripheral blood and spleen B cells
to polyvalent CR2 ligands on their [Ca"+]i.As such we
have used HB5 anti-CR2 antibody, F(ab']" fragments of
the same antibody, a synthetic tetravalent CR2 ligand:
the (P13),-template, and a pentavalent P28-BSA conjugate. Exposure of cells to intact HB5 for 5 min at 37°C
(or for up to 20 min) before addition of 3 pg/ml of the
F(ab'), fragment of anti-human p antibody clearly enhanced the increase of [Ca'+]i. The increase was dosedependent and the effect was reproduced in both positively and negatively selected peripheral blood (five experiments) and spleen B cells (two experiments). Optimal
enhancement wasobserved when suboptimal concentrations (3 pg/ml) of anti-p antibody were used. Because
cross-linking of B cell membrane Ig with the B cell FcR
inhibits B cell activation (22). we repeated this experiment with the F(ab')" fragment of HB5. Similar results
were obtained (Fig. 6). HB5 alonehad
no effect on
[Ca2+]i(Fig. 3). To determine if a tetravalent ligand bound
by CR2 would mimic the effect of the divalent HB5 antibody on the anti-p-induced increasein [Ca2+]i,
we studied
the ability of the synthetic, tetravalent
(P13),-template to
affect the increase. Preincubation of cells with (P13),template before addition of F(ab'), fragmentsof goat antihuman p enhanced the anti-p-induced [Ca2']i increase
(Fig. 7). As was observed with HB5. (P13),-template had
I
"0
1 0 4 20 30 5040
Time
FLgure4. The CR2-binding peptide P14 fails to inhibit the anti-p induced increase in [CaZ']i. Peripheral MNC were treated a s described in
the legend to Figure 2. After equilibration at 37°C the cells were treated
50 pm/ml; or (. . . . . . .). 100 Pg/ml of P14. or P14 (----)
with ,)-I
before exposure to 10 pg/ml of Flab'),-goat anti-p ( a r r o w ) .
1643
CR2 LIGANDS AND B CELL ACTIVATION
TABLE 11
Efiects o f P 2 8 a n d P28 conjugated to BSA on the anti-p-induced
increase in humanB cell [Ca2+]i
700
-2 600
= 500
C
.-c0
gC
400
Concentratlon of
F(ab'), Antl-p
Antlbody (pgfml)
h
No ligand
100 pglml P28
P28 conjugated to BSA (pglml)
10
396
50
100
Q
0
0
300
456
E
.g
-
3
10
231"
205
310
210
309
395
424
465
Maximum recorded channel [Ca2']i (nM); baseline [Caz+li was nM.
130
200
B cell enriched (76%)CD20') peripheral blood MNC were incubated with
100
the indicated ligands for 5 min before stimulation with the indicated
concentrations of anti-p antibody. P28or P28 conjugated to BSA did not
increase [Ca2+]iabove 140 nM.
9
TABLE III
HB-5 enhances anti-p-inducedE cell prollferation
0
cpm"
Figure 6. Effect of HB5 anti-CR2 antibody on the anti-p induced increase in B cell (Caz+]i. Peripheral
MNC were treated as described in the
(. . . . .). 0.5 (- - - -). or 1.O )-(
rgl
legend to Figure 2. treated with 0.1
ml of Flab'), fragment of HB5 or no HB5 (-.-.-.)
for 5 min, before
exposure to 3 pgfml of F(ab'), goat anti-p (arrow).
Sepharose-bound goat IgC"
Sepharose-bound
13,950
a-p
F(ab'),
Sepharose-bound HB5
+ SephaSepharose-bound F(ab')2 a-p
rose-bound HB5
Sepharose-bound HB5 and
F(ab')e goat-p
890 f 7 5
f 480
1.850 f 115
12,890 f 560
21,560 f 850
50,000 cells (75%CD20+)/200pi of medium/well were cultured in 96well plates, pulsed with 0.5 p Ci 3H-thymidine/weli for 18 h and harvested
after 72 hof culture.
All lg were bound to activated Sepharosea t a concentration of 1 mg/
ml of gel. Five pl of Sepharose-bound antibody wereadded to each ml of
culture medium.
a
4oot
I
TABLE IV
Effect ofC3d a n d P28 on anti-p antibody inducedB cell prollferation
cpm"
0
10 pglrnl F(ab'),
goat
anti-p
a
Time
Figure 7. Effect of (P13)4-template on the anti-p antibody induced
increase in B
cell [Caa+]i.Peripheral MNC were treatedas described in the
legend to Figure 2. After equilibrationa t 37°C they were treated with 10
for 5 min
pglml [- - - -), 50 pglrnl [ . . . . . .) or no (P14), template)-(
before adding3 pglmi of F(ab'), goat anti-p antibody.
no direct effect on B cell [Ca2']i (Fig. 3). Preincubation of
B cell-enriched human peripheral Bcells with P28bound
to BSA (molar ratio 5:l ) ,for 5 min before addition of antip antibody,alsoenhanced(in
three different experiments) the anti-p-induced increase in [Ca2']i (Table 11).
BSA alone had no effect on the anti-p-induced increase
in (Ca"+]i.
Finally, we asked whether theobserved effects of CR2
ligands on the anti-p antibody-induced increasein B cell
[Ca2']i were associated with similar effects
on anti-pinduced B cell proliferation. B cell-enriched peripheral
blood cells (70 to 80% CD20+ cells) were cultured with
Sepharose-bound F(ab'), fragments of anti-human p in
the presence or absence of HB5 antibody. The presence
of HB5, either in soluble form or coupled to Sepharose,
had no effect on the anti-p-induced proliferative response. In contrast, cultureof B cell-enriched PBMC with
Sepharose beads, to which both anti-p andHB5 antibod-
650 f 6 5
+O
8.750 k 470
+O
+10 pglml C3d
8.970 f 380
+50 pgfml C3d
5,480 f 350
+lo0 pglml C3d
2,350 f 115
f 350
+10 pglml P289.150
f 310
+50 pglml P285,730
+ l o 0 pgfml P281,250
f 65
Cultures wereperformed as described in Table11.
ies had been coupled, stimulated more DNA synthesis
than did Sepharose beads that bore anti-p, but not HB5
(TableHI). Culture of B cell-enriched peripheral blood cells
with soluble HB5 in the presence of 3 pg/ml of soluble
anti-p failed to induce detectable DNA synthesis, and
soluble HB5 had no effect on DNA synthesis induced by
10 pg/ml of soluble F(ab'I2 of anti-p. This was not surprising inasmucha s HB5 had little effect on the increase
of [Ca2']i that is stimulated by this concentration of antip antibody.
Monovalent CR2 ligands had a clear inhibitory effect
on the induction of B cell DNA synthesis by soluble F(ab'I2
fragments of anti-pantibody.
When B cell-enriched
PBMC were cultured with soluble F(ab'), anti-p antibody
in the presence of graded doses of P28 or C3d, B cell
proliferative responses were inhibited in a dose-dependent manner (Table IV). Thus, monovalent CR2 ligands
inhibit both the induction of increased B cell [Ca2+]iand
DNA synthesis by mitogenic concentrations of anti-p antibody, whereas divalent or polyvalent CR2 ligands enhance theability of submitogenic concentrations of antip antibody to stimulate a n increase in B cell [Ca2']i but
do not stimulate mitogenesis. Soluble divalent CR2 ligands have little effect on enhancement of B cell [Ca2']i
1644
CR2 LIGANDS AND B CELLACTIVATION
Ig, and because membrane Ig, whencross-linked, becomesassociatedwith
ligand-occupied CR2 (34), it is
possible that cross-linking of CR2 by divalent or polyvalent ligands promotes the formation
of domains of membrane Ig that have theconformation required to activate
phospholipase C. Such promotion of membrane Ig crosslinking by a n interaction between CR2 and a polyvalent
DlSCUSSlON
ligand would be particularly marked when the ligands for
In the present study we have used flow microfluori- membrane Ig and CR2 were physically associated. Notametry to study the effect of monovalent CR2 ligands or bly, we and others (15) have been best able to demonligands capable of cross-linking CR2 on the ability of strate synergistic induction of B cell DNA synthesis by
anti-p antibody to activate
B lymphocytes. Because of anti-Ig and anti-CR2 antibodies when thesetwo antibodvariability in earlier reports
of the effects of CR2 ligands ies have been directly associated. In contrast to its proon B cell proliferation and differentiation (1
1- 15, 28) we motion of membrane Ig cross-linking when occupied by
considered that the valency of the CR2 ligands might be a multivalent ligand, CR2, whenoccupied by a univalent
ligand, might act as a brake on the cross-linking of asimportantindeterminingtheCR2-transducedsignal.
sociated membrane Ig rather than contributeto its crossHence, we usedclearlydefinedmonovalent,divalent,
tetravalent, and pentavalentCR2 ligands. None of these linking. This could interfere with the establishment of
ligands had any effect on [Ca2'li if added alone at the cross-linked membrane Ig domains that are capable of
activating phospholipase C, with the direct result that
initiation of the analysis.
Occupancy of CR2 by monovalent ligands before stim- the increases in[Ca2+]iand protein kinase C activity are
ulation of B cells with anti-p antibody clearly inhibits B inhibited. This, in turncould limit the induction of a late
cell activation. In our study, monomeric C3d, a natural event inB cell activation, suchas DNA synthesis.
ligand of CR2, as well as the syntheticpeptide P28, which Regardless of the mechanism involved, the opposite
effects of CR2 interactions with monovalent or multivarepresents the CR2 bindingsequence(residues118712 14)
of C3, inhibited the anti-p induced[Ca"+]iincrease. lent ligands could have important physiologic effects on
the development of humoral immune responses in vivo.
P14, another synthetic peptide (residues 1201-1214) that
C the alternative pathway
also binds to CR2 but with lesser affinity (9).failed to Bacterial Ag that directly fix by
Ag-C3 fragmentcomplexesthat
inhibit significantly the anti-p-induced B cell fCa2']i re- would notonlyform
sponse. In previous reports, monomeric
CR2 ligands have could associate with multiple Ig and CR2 molecules on
been found to inhibit murine B cell progenitor [ 16) and the surfaceof Ag-specific B cells, but would also release
of the
lymphoblastoid B cell growth (10).Similarly, CR1 mono- soluble, univalent C3 fragments. The multivalence
a bacterium-C3 complex could
meric ligands inhibitB cell differentiation (29) and mac- C3 fragments on such
contribute to activation of B cells specific for bacterial
rophage Ia antigen expression (30).
In contrast to the effects of monovalent CR2 ligands, Ag, whereas univalent, circulating C3 fragments might
inhibit thepolyclonal activation of B cellsthat lack specpolyclonal anti-CR2 F(ab')2 (1 l ) , monoclonal anti-CR2
(12, 13) and glutaraldehyde bound
C3d (14) enhance anti-ificity for bacterialAg. Thus, theopposite effectsof monp antibody-induced B cell proliferation. Polymeric C3dg ovalent and polyvalent CR2 ligands on B cell activation
primes human B cells to proliferate more
vigorously upon could provide a means by which the C system could
of a specific
stimulation with anti-p antibody (31). In this study we simultaneously contribute to the generation
antibody
response
and
restrain
the
development
of a
used F(ab'), fragments of HB5 to demonstrate that they
enhance theability of suboptimal concentrationsof anti- polyclonal antibody response thatwould be drain on the
immune system and could produce potentially harmful
p antibody to induce an increase in[Ca"+]i.This finding
is in agreement with a recent report (15). We extended autoantibodies.
this observation by demonstrating thata synthetic tetraAcknowledgments. We aregratefulto
Dr. G. Thyvalent CR2 ligand ([P13),-template), and P28 conjugated
phronitis for his help with reagents, Mr. D. Smoot and
to BSA (pentavalent) similarly enhance the anti-p antiMr. D. Avila for expert technical assistance, and
Mrs.
body-induced increase in B cell [Ca2']i.
this manuscript.
Our study thus demonstrates that the valence of the Denise R. Giles for typing
CR2 ligand determines whether it inhibits or enhances
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