U.S. patent application number 08/954279 was filed with the patent office on 2002-01-10 for calcium-independent negative regulation by cd81 of receptor signalling.
Invention is credited to FLEMING, TONY, KINET, JEAN-PIERRE.
Application Number | 20020004210 08/954279 |
Document ID | / |
Family ID | 26709110 |
Filed Date | 2002-01-10 |
United States Patent
Application |
20020004210 |
Kind Code |
A1 |
FLEMING, TONY ; et
al. |
January 10, 2002 |
CALCIUM-INDEPENDENT NEGATIVE REGULATION BY CD81 OF RECEPTOR
SIGNALLING
Abstract
Calcium independent CD81 inhibition of IgE-mediated
degranulation in mast cells, particularly through the Fc.gamma.RIII
and Fc.epsilon.RI receptors, is described, as well as methods of
inhibiting allergic processes.
Inventors: |
FLEMING, TONY; (NEWTON,
MA) ; KINET, JEAN-PIERRE; (LEXINGTON, MA) |
Correspondence
Address: |
LISA M WARREN
HAMILTON BROOK SMITH & REYNOLDS
TWO MILITIA DRIVE
LEXINGTON
MA
021734799
|
Family ID: |
26709110 |
Appl. No.: |
08/954279 |
Filed: |
October 20, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60032963 |
Dec 13, 1996 |
|
|
|
Current U.S.
Class: |
435/7.21 ;
514/1.7; 514/20.6; 514/7.5 |
Current CPC
Class: |
C07K 16/2896 20130101;
C07K 14/70596 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/7.21 ; 514/2;
514/12 |
International
Class: |
A01N 037/18; A61K
038/00; G01N 033/567; A61K 039/395 |
Goverment Interests
[0002] Work described herein was funded by grant 1-RO1-GN53950-01
from the National Institutes of Health. The U.S. Government has
certain rights in the invention.
Claims
We claim:
1. A calcium independent method of inhibiting cell surface
receptor-mediated signaling comprising contacting a cell with an
agent which induces CD81-mediated signal transduction.
2. A method according to claim 1, wherein the cell surface receptor
is selected from the group consisting of Fc.epsilon.RI and
Fc.gamma.RIII .
3. A calcium-independent method of inhibiting degranulation
comprising contacting a cell with an agent which induces
CD81-mediated signal transduction.
4. A method according to claim 3, wherein the degranulation is
mediated by the Fc.epsilon.RI receptor.
5. A calcium independent method of inhibiting cell surface
receptor-mediated signaling in a mammal comprising administering to
the mammal an effective amount of an agent which induces
CD81-mediated signal transduction.
6. A method according to claim 5, wherein the cell surface receptor
is selected from the group consisting of Fc.epsilon.RI and
Fc.gamma.RIII .
7. A calcium independent method of inhibiting degranulation induced
by a cell surface receptor-mediated signal in a mammal comprising
administering to the mammal an effective amount of an agent which
induces CD81-mediated signal transduction.
8. A method of treating an allergic condition in a mammal
comprising administering to the mammal an effective amount of an
agent which induces CD81-mediated signal transduction.
9. A method according to claim 8, wherein the allergic condition is
asthma, hay fever or atopic eczema.
10. A calcium independent method of enhancing cell surface
receptor-mediated signaling comprising contacting a cell with an
agent which inhibits CD81-mediated signal transduction.
11. A method according to claim 10, wherein the cell surface
receptor is selected from the group consisting of Fc.epsilon.RI and
Fc.gamma.RIII .
12. A calcium independent method of enhancing degranulation
comprising contacting a cell with an agent which inhibits
CD81-mediated signal transduction.
13. A method according to claim 12, wherein the degranulation is
mediated by the Fc.epsilon.RI receptor.
14. A calcium independent method of enhancing cell surface
receptor-mediated signaling in a mammal comprising administering to
the mammal an effective amount of an agent which inhibits
CD81-mediated signal transduction.
15. A method according to claim 14, wherein the cell surface
receptor is selected from the group consisting of Fc.epsilon.RI and
Fc.gamma.RIII .
16. An assay for identifying agents which alter CD81-mediated
signal transduction, comprising the steps of: a) combining a cell
bearing CD81 with an agent to be tested under conditions suitable
for CD81-mediated signal transduction; and b) determining the level
of CD81-mediated signal transduction, wherein if the level of
CD81-mediated signal transduction is altered relative to a control,
the agent alters CD81-mediated signal transduction.
17. An assay for identifying agents which alter calcium independent
CD81-mediated regulation of cell surface receptor signaling,
comprising the steps of: a) combining a cell bearing CD81 and an
appropriate cell surface receptor with an agent which alters
CD81-mediated signal transduction under conditions suitable for
signal transduction by CD81 and the cell surface receptor; and b)
determining the level of cell surface receptor signaling; wherein
if the level of cell surface receptor signaling is altered relative
to a control, the agent alters calcium independent CD81-mediated
regulation of cell surface receptor signaling.
18. A method according to claim 17, wherein the cell surface
receptor is selected from the group consisting of Fc.epsilon.RI and
Fc.gamma.RIII .
19. A method of inhibiting passive cutaneous anaphylaxis in a
mammal comprising administering to the mammal an effective amount
of an agent which enhances CD81-mediated signal transduction.
20. A method according to claim 19, wherein the agent is an
anti-CD81 monoclonal antibody.
Description
RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Serial No. 60/032,963, filed Dec. 13, 1996, the entire
teachings of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] In the past two decades, tremendous advances have been made
in understanding the molecular mechanisms used by various types of
cell surface receptors to transduce signals. Nearly all of these
advances have come from the study of model systems where a receptor
"activates" cells to generate a well-defined response. As knowledge
about activating model systems has increased, it has become clear
that there are many situations in which the activating signal sent
from one receptor is modulated as the direct result of a negative
or inhibitory signal sent by another cell surface receptor. While
the study of this type of signaling is generally in its infancy,
several recent studies have begun to shed light on the molecular
mechanisms which underlie receptor-mediated inhibitory signals in
immunologic systems. Given the tendency of nature to utilize
signaling functions modularly in a variety of signaling pathways,
the paradigms outlined by these systems may have implications for
the study of inhibitory or deactivating signals in non-immunologic
situations as well. In addition, the study of these signals may add
new dimensions to the understanding of other widely utilized
signaling pathways.
SUMMARY OF THE INVENTION
[0004] As described herein, monoclonal antibodies (mAbs) have been
isolated which inhibit Fc.epsilon.RI-induced mast cell
degranulation. Through protein isolation, peptide sequencing,
cloning, and gene expression, CD81 has been identified as a novel
inhibitory receptor for Fc.epsilon.RI and Fc.gamma.RIII. Anti-CD81
mAbs also inhibited passive cutaneous anaphylaxis (PCA) reactions,
a model of IgE-dependent, mast cell activation in vivo.
[0005] The invention pertains to a method of inhibiting cell
surface receptor-mediated signaling comprising contacting a cell
with an agent which induces CD81-mediated signal transduction. In a
particular embodiment, the cell surface receptor is selected from
the group consisting of Fc.epsilon.RI and Fc.gamma.RIII. In one
embodiment, the method is a calcium independent method.
[0006] The invention also relates to a method of inhibiting
degranulation comprising contacting a cell with an agent which
induces CD81-mediated signal transduction. In one embodiment,
degranulation is mediated by the Fc.epsilon.RI receptor. In another
embodiment, the method is a calcium independent method.
[0007] The invention further relates to a calcium independent
method of inhibiting cell surface receptor-mediated signaling in a
mammal, such as a human, comprising administering to the mammal an
effective amount of an agent which induces CD81-mediated signal
transduction. In one embodiment, the cell surface receptor is
selected from the group consisting of Fc.epsilon.RI and
Fc.gamma.RIII.
[0008] The invention also pertains to a method, e.g., a calcium
independent method, of inhibiting degranulation induced by a cell
surface receptor-mediated signal in a mammal, such as a human,
comprising administering to the mammal an effective amount of an
agent which induces CD81-mediated signal transduction.
[0009] The invention further pertains to a method of treating
(e.g., preventing or reducing the severity of) an allergic
condition in a mammal, such as a human, comprising administering to
the mammal an effective amount of an agent which induces
CD81-mediated signal transduction. In particular embodiments, the
allergic condition is asthma, hay fever or atopic eczema.
[0010] The invention also relates to a calcium independent method
of enhancing cell surface receptor-mediated signaling, e.g.,
Fc.epsilon.RI-mediated signaling and Fc.gamma.RIII-mediated
signaling, comprising contacting a cell with an agent which
inhibits CD81-mediated signal transduction.
[0011] The invention also pertains to a calcium-independent method
of enhancing degranulation comprising contacting a cell with an
agent which inhibits CD81-mediated signal transduction. For
example, degranulation can be mediated by the Fc.epsilon.RI
receptor. The invention also relates to a calcium independent
method of enhancing cell surface receptor-mediated signaling in a
mammal comprising administering to the mammal an effective amount
of an agent which inhibits CD81-mediated signal transduction.
[0012] The invention further relates to an assay for identifying
agents which alter CD81-mediated signal transduction, comprising
combining a cell bearing CD81 with an agent to be tested, under
conditions suitable for CD81-mediated signal transduction, and
determining the level of CD81-mediated signal transduction. If the
level of CD81-mediated signal transduction is altered relative to a
control, the agent alters CD81-mediated signal transduction. In a
particular embodiment, the agent is one which enhances or induces
CD81-mediated signal transduction.
[0013] The invention also relates to an assay for identifying
agents which alter calcium independent CD81-mediated regulation of
cell surface receptor signaling, comprising combining a cell
bearing CD81 and an appropriate cell surface receptor with an agent
which alters CD81-mediated signal transduction under conditions
suitable for signal transduction by CD81 and the cell surface
receptor, and determining the level of cell surface receptor
signaling. If the level of cell surface receptor signaling is
altered relative to a control, the agent alters calcium independent
CD81-mediated regulation of cell surface receptor signaling.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A-1C illustrate representative immunologic inhibitory
signaling systems. Solid dots on sIg, Fc.gamma.RIII and TCR
indicate tyrosine phosphorylation of activating motifs in the
cytoplasmic tails of each activating receptor (FIGS. 1A-1C,
respectively). Solid dots on Fc.epsilon.RIIB, KIR and CTLA-4
indicate tyrosine phosphorylation of inhibitory motifs in the
cytoplasmic tails of each inhibitory receptor (FIGS. 1A-1C,
respectively). FIG. 1A illustrates the surface immunoglobulin
receptor (sIg) complex and Fc.gamma.RIIb1 system. Fc.gamma.RIIb1
provides a negative feedback signal for soluble immunoglobulin
production. FIG. 1B illustrates the negative regulation of
cytolytic immune cells by killer cell inhibitory receptors (KIR).
FIG. 1C illustrates the negative regulation of T-cell
receptor-mediated activation signals by CTLA-4.
[0015] FIG. 2 illustrates the schematic structures of SHP1, SHP2
and SHIP.
[0016] FIG. 3 illustrates the proposed SHP and SHIP inhibitory
signaling mechanisms. Solid dots on sIg indicate tyrosine
phosphorylation of activating motifs in the cytoplasmic tails of
each activating receptor. Solid dots indicate tyrosine
phosphorylation of inhibitory motifs in the cytoplasmic tails of
each inhibitory receptor.
[0017] FIG. 4 illustrates 5D1 mAb inhibition of
Fc.epsilon.RI-mediated degranulation in RBL-2H3 cells.
[0018] FIG. 5 illustrates Ly-C peptide 1A12 sequence and alignment
with mouse and human CD81.
[0019] FIGS. 6A-6B are the results of FACS analysis illustrating
expression of rat CD81 in CHO arid NIH-3T3 cells. FIG. 6A shows
stable expression of rat CD81 in CHO cells stained with 1A12 mAb.
FIG. 6B shows transient expression of rat CD81 in NIH-3T3 cells
infected with M.O.I.=5 of rat CD81 recombinant vaccinia virus and
incubated for 6 hours prior to staining with 5D1 mAb.
[0020] FIGS. 7A-7D are graphs of the effect of preincubation of
purified mAb 5D1 on Fc.epsilon.RI-mediated degranulation in RBL-2H3
cells. Data shown indicate the results of degranulation of
IgE-saturated RBL-2H3 cells after incubation with buffer (filled
circles) or purified 5D1 mAb at 2.5 ng (filled squares), 25 ng
(filled triangles), or 250 ng (filled inverted triangles) (FIGS.
7A, 7C, 7D) or with 100 ng (7B) of 5D1 mAb per 10.sup.5 cells prior
to triggering with the indicated concentrations of DNP-HSA (FIG.
7A), 50 ng/ml DNP-HSA (FIGS. 7C and 7D) or with PMA and ionomycin
(FIG. 7B). Data are expressed as mean dpm .+-.standard deviation or
as percentages of control (no antibody) mean dpm. Statistical
significance versus untreated controls was determined using an
unpaired Student's t-test: *, p<0.05; **, p<0.01; ***,
p<0.001 for FIG. 8A. All data points in FIGS. 8B and 8D were
found to be significantly different from controls (p<0.02) with
the exception of the 5 minute preincubation time point with 2.5 ng
mAb 5D1 (FIG. 8C, p=0.067).
[0021] FIG. 8 shows expression of rat CD81 in mouse mast cell line
C1.MC/C57.1 by FACS staining with 5D1 and 1A12 mAbs.
[0022] FIGS. 9A-9C are graphs showing that CD81 mAbs fail to
inhibit Fc.epsilon.RI-induced tyrosine phosphorylation, calcium
mobilization, and leukotriene synthesis. FIG. 9A shows the effect
of anti-CD81 on calcium mobilization of fura-2-loaded RBL-2H3 cells
triggered through Fc.epsilon.RI as measured by confocal microscopy.
Fluo-3 fluorescence per ml .sup.3H measurements were normalized by
dividing the average fluorescence intensity (F) occurring during
the course of the experiment to the average fluorescence intensity
at the beginning of the experiment (F.sub.0) and expressed as
F/F.sub.0. Traces are shown of 10 individual cell (thin lines)
together with mean values for these cells (thick lines) and
represent typical results obtained from five separate experiments.
FIG. 9B shows .sup.3H-serotonin release from RBL-2H3 cells prepared
as in confocal microscopy measurements except that 3 .mu.Ci/ml
.sup.3H-serotonin was added to cultures. FIG. 9C shows LTC.sub.4
measurements from 106 anti-DNP IgE saturated RBL-2H3 treated with 1
.mu.g 5D1 (open squares) or buffer (open circles) prior to
triggering with 30 ng/ml DNP-HSA for the indicated periods of
time.
[0023] FIG. 10A-10B are graphs showing inhibition of passive
cutaneous anaphylaxis in Wistar rats by anti-CD81. Male Wistar rats
were injected with (FIG. 10A) 25 ng DNP-specific IgE mixed with 50
.mu.g anti-CD81 mAb 5D1 (mouse IgG1) or control mouse IgG1 mAb
(MOPC 31c, specificity unknown) or (FIG. 10B) 100 ng DNP-specific
IgE alone. Statistical significance was determined using an
unpaired Student's t-test: *, p<0.05; **, p<0.01(actual
values 10A, p=0.024 versus MOPC 31c controls; 10B, p=0.009 versus
anti-LFA-1.beta. controls).
[0024] FIGS. 11A-11D are the results of FACS analysis of 3 stable
mouse Fc.gamma.RIII RBL-2H3 transfectants after staining with 2.4G2
and FITC-anti-rat IgG.
[0025] FIG. 12 is a set of graphs illustrating that DNP-HSA induces
IgE-mediated degranulation in four different cell lines and that
this degranulation is inhibitable by anti-CD81 mAb 5D1.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Mast cells are important effector cells in IgE-dependent
immune responses and allergic diseases (Galli, New. Engl. J. Med.
328:257-265 (1993)), and mast cells also contribute to host defense
against parasites and bacteria (Echtenacher et al., Nature
381:75-77 (1996), Galli and Wershil, Nature 381:21-22 (1996)).
Crosslinking of FC.epsilon.RI-IgE complexes on mast cells and
basophils by multivalent antigen initiates a signaling cascade
characterized by tyrosine kinase activation, calcium release and
influx and, later, by degranulation and release of inflammatory
mediators (Jouvin et al., J. Biol. Chem. 269:5918-5925 (1994);
Penhallow et al., J. Biol. Chem. 270:23362-23365 (1995);
Scharenberg et al., EMBO J. 14:3385-3394 (1995); Lin et al., Cell
85:985-995 (1996); and (Paul et al., Adv. Immunol. 53:1-29
(1993)).
[0027] Like the B and T cell antigen receptors, Fc.gamma.RI lacks
endogenous signaling capacity and utilizes tyrosine phosphorylation
to recruit signaling effector molecules. Receptor aggregation leads
to phosphorylation and/or activation of several protein tyrosine
kinases (PTKs) Lyn, Syk, Btk, Itk, Fer, and FAK (Jouvin et al., J.
Biol. Chem. 269:5918-5925 (1994); Penhallow et al., J. Biol. Chem.
270:23362-23365 (1995); Scharenberg et al., EMBO J. 14:3385-3394
(1995); and Kawakami et al., Mol. Cell. Biol. 14:51014:5108 -5113
(1994); Kawakami et al. , J. Immunol. 155:3556-3562 (1995); and
Hamawy et al., J. Biol. Chem. 268:6851-6854 (1993)), as well as
protein kinase C isoenzymes (Ozawa et al., J. Biol. Chem.
268:1749-1756 (1993)) , MAP kinase (Hirasawa et al., J. Biol. Chem.
270:10960-10967 (1995)), and other signaling molecules such as Cbl
and Shc (Ota et al., J. Exp. Med. 184:1713-1723 (1996); and
Jabril-Cuenod et al., J. Biol. Chem. 271:16268-16272 (1996)).
[0028] The precise role of many of these proteins in degranulation
remains undefined. However, it is clear that Fc.epsilon.RI-mediated
calcium mobilization, degranulation, and leukotriene and cytokine
synthesis depend on early tyrosine kinase activation events,
especially the activation of the PTK Syk. Fc.epsilon.RI signaling
is initiated by tyrosine phosphorylation of immunoreceptor
tyrosine-based activation motifs (ITAM; defined by the sequence
(D/E)x.sub.xYx.sub.2Lx.sub.6-7Yx.su- b.2(L/I) (Flaswinkel et al.,
Semin. Immunol 7:21-27 (1995)). Phosphorylated ITAMs (pITAMs)
facilitate binding of SH2-domain-containing proteins to
Fc.epsilon.RI (Johnson et al., J. Immunol. 155:4596-4603 (1995);
Kimura et al., J. Biol. Chem. 271:27962-27968 (1996)).
[0029] In addition to activation events, receptor-activated PTKs
initiate the regulation of antigen receptor signaling by
phosphorylating tyrosine-based motifs on membrane receptors known
as inhibitory receptors (Scharenberg and Kinet, Cell 87:961-964
(1996); Cambier, Proc. Natl. Acad. Sci. USA 94:5993-5995 (1997)).
These proteins bind SH2-domain-containing phosphatases, the
tyrosine phosphatases SHP-1 and SHP-2 and the phosphatidylinositol
(Scharenberg et al., EMBO J. 14:3385-3394 (1995); Lin et al. , Cell
85:985-995 (1996); Paul et al., Adv. Immunol. 53:1-29 (1993)) 5'
phosphatase SHIP, upon coengagement with antigen or growth factor
receptors. Although the molecular targets are still being defined,
phosphatase recruitment to inhibitory receptors has one of two
general effects on signaling. Engagement of inhibitory receptors
that preferentially bind SHIP, such as the low affinity receptor
for IgG (Fc.gamma.RIIbl) (Ono et al., Nature 383:263-266 (1996))
results in selective inhibition of calcium influx with little or no
effect on receptor-mediated calcium release or tyrosine
phosphorylation. On the other hand, killer cell inhibitory
receptors (KIR) bind SHP-1 upon receptor costimulation, resulting
in reduced tyrosine phosphorylation, calcium release from the ER,
and calcium influx (Burshtyn et al., Immunity 4:77-85 (1996);
Binstadt et al., Immunity 5:629-638 (1996)). In both mechanisms,
calcium mobilization is inhibited along with downstream signaling
events.
[0030] Descriptions of three representative Systems utilized in
recent studies are useful for understanding the nature of
inhibitory signals, and are outlined in FIGS. 1A-1C. Briefly, the
surface immunoglobulin receptor (sIg) complex and Fc.gamma.RIIb1 (a
low affinity receptor for IgG) are both normally present on B-cell
surfaces (FIG. 1A, left panel). When sIg receptors are clustered as
a result of contact with antigen (FIG. 1A, middle panel), they
typically produce a cell activation signal which induces B-cell
proliferation. However, if the same B-cells are stimulated so that
the sIg receptors are co-clustered with Fc.gamma.RIIb1 receptors
(for example by contact of the B-cell with an immune complex of
cognate antigen and IgG, FIG. 1A, right panel), B-cells fail to
proliferate and in some cases may apoptose.
[0031] In the natural killer (NK) cell system, a number of cell
surface receptors are able to initiate NK cell cytolysis, one of
which is Fc.gamma.RIII (FIG. 1B, left panel). When an NK cell
encounters a target cell, it recognizes and kills the target cell
if the target cell lacks class I MHC molecules. One of the ways in
which NK cells recognize target cells is by binding of IgG bound to
the target cell surface to Fc.gamma.RIII on NK-cells (FIG. 1B,
middle panel). If the target cells express appropriate class I MHC
molecules which can be recognized by appropriate killer cell
inhibitory receptors (KIR) on the NK cell, they are protected from
cytolysis (FIG. 1C, right panel).
[0032] In the T-cell system, the T-cell antigen receptor (TCR) and
CD4 and/or CD8 co-receptors are normally expressed on the surface
of resting T-cells (FIG. 1C, left panel). T-cells are activated
when their T-cell antigen receptor complexes (TCR's) interact with
specific peptide/MHC class II complexes on antigen presenting cells
(APCs), resulting in co-clustering of the TCR and CD4 or CD8 (FIG.
1C, middle panel). Upon activation, T-lymphocytes upregulate
expression of another surface molecule called CTLA-4, which results
in interaction of CTLA-4 with its countereceptors CD80 or CD86
(FIG. 1C, right panel). Since mice which lack CTLA-4 have
hyperactivated T-cells and are prone to lymphoproliferative
diseases, it is thought that CTLA-4 mediates an inhibitory signal
which provides an important negative feedback control for
proliferation and cytokine production induced by T-cell receptor
activation signals.
[0033] While each of these systems is unique in terms of the manner
in which the activating and inhibitory signals are engaged, two
common features exist among them: 1) Each involves activating
signals mediated by homologous cytoplasmic tail motifs known as
immunoreceptor tyrosine based activation motifs (ITAMs). These
motifs become tyrosine phosphorylated by src family kinases when
the activating receptors are engaged by clustering stimuli,
resulting in the recruitment to engaged receptors of both src and
syk/zap70 family non-receptor tyrosine kinases. Downstream
propagation of the activation signal is then mediated by activation
of these tyrosine kinases and the resulting phosphorylation of
specific substrates. 2) The inhibitory signals are mediated by
separate receptors, such as Fc.gamma.RIIb1, killer cell inhibitory
receptors (KIR), and CTLA-4, which are engaged in concert with the
activating receptor when an appropriate stimulus is present. When
the inhibitory receptors are appropriately engaged, they become
phosphorylated on specific cytoplasmic tail tyrosines by src family
kinases, which results in the recruitment of signaling molecules
which are inhibitory in function.
[0034] It appears that SHP-1/SHP-2 and SHIP are recruited for
distinct purposes. SHP-1 and SHP-2 attenuate or completely block
tyrosine phosphorylation-mediated signals (FIG. 3, middle panel),
while SHIP allows a full strength tyrosine phosphorylation signal
to proceed while blocking any downstream events which require
sustained elevations of soluble inositol phosphates and/or
intracellular calcium (FIG. 3, right panel). One potential
explanation can be rationalized by comparing the function of the
inhibitory signals mediated by Fc.gamma.RIIb1 on B-cells and KIR on
NK cells. The sIg receptor activating signal serves to notify
B-cells that specific antigen is present, and so initiate B-cell
maturation and proliferation for the purpose of specific
immunoglobulin production. However, coengagement of sIg and
Fc.gamma.RIIb1 blocks proliferation and can induce apoptosis of the
B-cell and a consequent decrease in production of specific
immunoglobulin, thereby acting as a negative feedback mechanism.
Thus, it appears that the persistence of a full strength tyrosine
phosphorylation signal in the absence of sustained inositol
phosphate and/or intracellular calcium levels is for the purpose of
notifying the B-cell that adequate specific antibody has been
produced, and may be the signal which induces apoptosis of that
B-cell in the appropriate context.
[0035] This situation is subtly, but importantly, different than
that of an NK cell. NK cells function by undergoing target cell
recognition events mediated by activating receptors which are
capable of initiating cytolysis, and the KIR inhibitory signal is
required to block inappropriate cytolysis of cells which are
recognized but which also bear appropriate class I MHC. Since there
would be little utility in the NK cell "knowing" about contact with
each and every protected target, an inhibitory mechanism where the
activating signal is completely abrogated would seem to be most
appropriate. This would account for the apparently SHP-1
predominant inhibitory signal mediated by KIR. To summarize, these
results suggest that primarily SHP-l/SHP-2 mediated block would be
utilized when the cell has no need to know about the presence of a
particular stimulus, while a primarily SHIP-mediated block would be
utilized when the cell needs to know and to respond in some altered
manner.
[0036] IgE-dependent activation of mast cells primarily occurs
through antigen-mediated crosslinking of IgE-Fc.gamma.RI complexes
which initiates a signaling cascade ultimately leading to release
of proinflammatory mediators (Scharenberg and Kinet, Chem. Immunol.
61:72-87 (1995)). Fc.gamma.RI is a member of the multi-subunit,
antigen receptor family which includes B and T cell receptors (BCR
and TCR) and receptors for the Fc portions of IgA and IgG (Ravetch
and Kinet, Ann. Rev. Immunol. 9:457-492 (1991)). These receptors
share common features of immunoglobulin-like ligand binding
subunit(s) and associated signaling polypeptides which lack
endogenous enzymatic activity.
[0037] In mast cells, both Fc.epsilon.RI and Fc.gamma.RIII are
expressed as .alpha..beta..gamma..sub.2 tetramers in which the
respective .beta. and FcR.gamma. signaling chains are identical and
the ligand-binding .alpha.chains are different. In Fc.epsilon.RI,
the high affinity IgE binding domain is localized to the
Fc.epsilon.RI.alpha. subunit (Blank et al., J. Biol. Chem.
266:2639-2646 (1991)) and IgE binding to Fc.epsilon.RI.alpha.
itself does not contribute to signaling. The Fc.epsilon.RI.beta.
chain and the FcRg homodimer are the signaling components of the
Fc.epsilon.RI (.alpha..beta..gamma.2) tetrameric receptor. Both
Fc.epsilon.RI.beta. and FcR.gamma. have one copy per chain of the
immunoreceptor tyrosine-based activation motif (ITAM; Flaswinkel et
al., Semin. Immunol. 7:21-27 (1995), Cambier, J. Immunol.
155:3281-3285 (1995)) defined by the sequence Yx2Lx6-7Yx2L/I.
[0038] Fc.epsilon.RI signaling is an aggregation-dependent
phenomenon in which multivalent antigen crosslinking of
IgE-Fc.epsilon.RI complexes initiates a signaling cascade ITAM
tyrosine phosphorylation by src family kinases (Shaw et al., Semin.
Immunol. 7:13-20 (1995)). Signaling through Fc.epsilon.RI is
characterized initially by tyrosine phosphorylation of
Fc.epsilon.RI.beta. and FcR.gamma. ITAMs by the .beta.-associated
src family kinase lyn (Jouvin et al., J. Biol. Chem. 269:5918-5925
(1994)). The lyn-phosphorylated ITAM (pITAM) interaction results in
lyn activation. Direct binding of lyn to fusion proteins containing
the Fc.epsilon.RI.beta., but not the FcR.gamma. ITAM, has been
demonstrated (Jouvin et al., J. Biol. Chem. 269:5918-5925 (1994)).
pITAM peptides have been shown to induce lyn phosphorylation both
in permeabilized cells and in vitro (Johnson et al., J. Immunol.
155:4596-4603 (1995)).
[0039] Following lyn activation, syk is recruited to FcR.gamma.
pITAMs via its SH2 domains where it is phosphorylated and activated
(Scharenberg and Kinet, Chem. Immunol. 61:72-87 (1995); Jouvin et
al., J. Biol. Chem. 269:5918-5925 (1994)). FcR.gamma..beta. pITAM
peptides were much more effective than Fc.epsilon.RI.beta. pITAM
peptides at activating syk in vitro in unstimulated RBL-2H3 lysates
(Shiue et al., J. Biol. Chem. 270:10498-10502 (1995)). Activated
lyn and syk phosphorylate a number of intracellular substrates
including PLC.gamma.1, BTK, ITK and cbl (Rawlings et al., Science
271:822-825 (1996); Kawakami et al., J. Immunol. 155:3556-3562
(1995)). Following initial tyrosine kinase activation events,
Fc.epsilon.RI signaling, like that of other antigen receptors,
involves calcium release from the endoplasmic reticulum (tyrosine
kinase-dependent) and a calcium influx, both of which precede
degranulation and the release of preformed mediators by granule
fusion with the cytoplasmic membrane. An interesting difference
between Fc.epsilon.RI and other antigen receptors is that calcium
mobilization through Fc.epsilon.RI appears to utilize sphingosine
kinase and sphingosine-1-phosphate (S-1-P) (Choi et al., Nature
380:634-636 (1996)) as opposed to the classical phospholipase
C/InsP3 pathway.
[0040] The rat basophilic leukemia cell line, RBL-2H3, has been
widely employed as a model cell in the study of
Fc.epsilon.RI-mediated activation. There have been a few reports of
monoclonal antibodies (mabs) directed to membrane components in
which co-ligation inhibits Fc.epsilon.RI-mediated degranulation in
mast cells. The best characterized examples are MAFA (mast cell
function-associated antigen) (Guthmann et al., Proc. Natl. Acad.
Sci. USA 92:9397-9401 (1995)) and gp49b1 (Katz et al., Proc. Natl.
Acad. Sci. USA 93:10809-10814 (1996)). MAFA is an Mr 20 kd C-type
lectin expressed in RBL-2H3 cells both as a monomer and
disulphide-linked homodimer that inhibits degranulation by acting
upstream of Fc.epsilon.RI-mediated activation of phospholipase Cg1
activation by tyrosine kinases (Guthmann et al., Proc. Natl. Acad.
Sci. USA 92:9397-9401 (1995)).
[0041] The target of gp49B1 is less well defined; however it
appears to act via a tyrosine-based ITIM (immunoreceptor
tyrosine-based inhibitory motif) defined by the sequence
V/Ix2Yx2I/L utilized by the NK inhibitory receptor (KIR), CD22,
CTLA-4, and Fc.gamma.RII.beta.1. Tyrosine phosphorylation of the
ITIM in KIR induces binding of the SHP-1 tyrosine phosphatase.
SHP-1 recruitment is intimately associated with inhibition of
calcium influx and mobilization presumably enacted through
yet-to-defined dephosphorylation events. Overexpression of
phosphatase-inactive SHP-1 ablates the inhibitory activity of
endogenous SHP-1. ITIM-mediated recruitment is not restricted to
SHP-1, as a second SH2-containing phosphatase (SHP-2) is utilized
by CTLA-4, and the FcgRIIb1 ITIM binds either SHP-1 or the
SH2-containing inositol phosphatase (SHIP). In the case of gp49b1,
it is unclear which effector is being utilized but it has been
demonstrated that a splice variant (gp49A) which lacks the
cytoplasmic ITIM but is identical in the extracellular domains
lacks detectable inhibitory activity. In addition to MAFA,
antibodies to the glycolipid Gd1b and the AD1 antigen (rat
homologue of CD63) have also been described to inhibit
Fc.epsilon.RI-mediated degranulation in RBL-2H3 cells.
[0042] Clustering of the high affinity IgE receptor (FC.epsilon.RI)
by antigen initiates a signaling cascade characterized by tyrosine
kinase activation, calcium release and influx and, later, by
degranulation and release of inflammatory mediators. In order to
examine how Fc.epsilon.RI signaling is negatively regulated, a
panel of monoclonal antibodies to mast cell membrane antigens was
generated and screened for inhibition of IgE-mediated mast cell
degranulation. Two degranulation inhibitory antibodies, designated
1A12 and 5D1, immunoprecipitated a Mr 25 kd protein from
surface-iodinated rat basophilic leukemia (RBL-2H3) cells. Lys-C
peptide sequence obtained from 1A12-immunoaffinity purified
immunoprecipitates was found to be highly homologous to mouse and
human CD81. Subsequent cloning and expression of rat CD81 cDNA from
RBL-2H3 confirmed that 1A12 and 5D1 recognize rat CD81 and that
CD81 crosslinking inhibits Fc.epsilon.RI-mediated mast cell
degranulation.
[0043] Signaling through the high affinity receptor for
immunoglobulin E (Fc.epsilon.RI) results in the coordinate
activation of tyrosine kinases prior to calcium mobilization.
Receptors capable of interfering with the signaling of antigen
receptors, such as Fc.epsilon.RI, recruit tyrosine and inositol
phosphatases that results in diminished calcium mobilization. It is
shown herein that antibodies recognizing CD81 inhibit
Fc.epsilon.RI-mediated mast cell degranulation but, surprisingly,
without affecting aggregation-dependent tyrosine phosphorylation,
calcium mobilization, or leukotriene synthesis. Furthermore, CD81
antibodies also inhibit mast cell degranulation in vivo as measured
by reduced passive cutaneous anaphylaxis responses. These results
reveal an unsuspected calcium-independent pathway of antigen
receptor regulation which is accessible to engagement by membrane
proteins and on which novel therapeutic approaches to allergic
diseases can be based.
[0044] CD81 belongs to the transmembrane 4 superfamily (TM4SF)
which includes CD9, CD53, CD63 and CD82 (Wright and Tomlinson,
Immunol. Today 15:588-594 (1994)). TM4SF proteins have been found
to associate with HLA-DR, CD4, CD19/21/Leu-13, small GTP-binding
proteins and an unidentified tyrosine phosphatase and (via mAb
crosslinking) to induce calcium mobilization and activate syk.
[0045] CD81 is broadly expressed on hematopoietic cells (T and B
lymphocytes, granulocytes, monocytes) and on some non-lymphoid
tumors. The function of CD81 (or other TM4SF proteins) is
incompletely understood, although CD81 appears to modulate the
signaling of other membrane receptors. CD81 is found in the
CD19/CD21 complex on B cells, and mAbs to CD81 or CD19 have been
reported to reduce the threshold for B cell receptor signaling
(Fearon and Carter, Annu. Rev. Immunol. 13:127-149 (1995)) and
enhance B cell adhesion via VLA4 (Behr and Schriever, J. Exp. Med.
182:1191-1199 (1995)). Consistent with a costimulatory role in B
cell receptor signaling, CD81 -/- mice express lower levels of CD19
on B cells which is proposed to contribute to a defect in humoral
immunity (Maecker and Levy, J. Exp. Med. 185:1505-1510 (1997)). For
T lineage cells, both stimulatory and inhibitory activities for
anti-CD81 mAbs have been reported (Secrist et al.,, Eur. J.
Immunol. 26:1435-1442 (1996); Todd et al., J. Exp. Med.
184:2055-2060 (1996); Oren et al., Mol. Cell. Biol. 10:4007-4015
(1990); and Boismenu et al., Science 271:198-200 (1996)). CD81
ligation enhances IL-4 production from antigen-specific CD4+T cells
(Secrist et al, Eur. J. Immunol. 26:1435-1442 (1996)) and integrin
activation and IL 2-dependent proliferation in human thymocytes
(Todd et al., J. Exp. Med. 184:2055-2060 (1996)). Alternatively,
CD81 was originally called TAPA-1 (target of antiproliferative
antibody) based on inhibition of proliferation in human T cell
lines by CD81 antibodies (Oren et al., Mol. Cell. Biol.
10:4007-4015 (1990)). Some of these pleiotropic effects may stem
from the potential signaling molecules with which CD81 has been
reported to associate including CD4, CD8, MHC class II, other TM4SF
proteins, integrin VLA4, and phosphatidylinositol 4-kinase (Wright
and Tomlinson, Immunol. Today 15:588-594 (1994); Imai et al., J.
Immunol. 155:1229-1239 (1995); Angelisova et al., Immunogenetics
39:249-256 (1994); Mannion et al., J. Immunol. 157:2039-2047
(1996); and Berditchevski et al., J. Biol. Chem. 272:2595-2598
(1997)).
[0046] Mast cell Fc.epsilon.RI can be saturated with monoclonal IgE
antibodies. In the absence of crosslinking by appropriate antigen,
IgE binding to Fc.epsilon.RI does not activate mast cells.
Monoclonal antibodies are purified from culture supernatants or
mouse ascitic fluid (produced by injection of antibody-producing
cells into immunocompromised mice by standard techniques, such as
those described in Kohler and Milstein, Nature 256:495-497 (1975);
Kozbar et al., Immunology Today 4:72 (1983); and Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96 (1985)). Crosslinking by the antigen (protein binding to the
IgE) normally induces cell degranulation which can be quantitated
by enzyme assay or radioactivity release assay. Antibody treatment
of CD81 mast cells inhibits IgE-mediated degranulation; 20 ng of
5D1 monoclonal antibody per 10.sup.5 RBL-2H3 cells inhibits
degranulation through IgE-mediated channels by greater than
75%.
[0047] Mast cells are a major cell in allergic reactions. Thus, the
present invention can be used to develop agents, e.g., antibodies,
which inhibit the allergic process, as well as to develop compounds
for the treatment of allergies, anaphylactic reactions and related
diseases. Agents can also be developed which mimic the process of
CD81-mediated inhibition of mast cell degranulation. Anti-CD81
antibodies are more inhibitory than antibodies to other different
proteins for IgE-mediated degranulation, particularly because
anti-CDb 81 antibodies act directly and do not require secondary
reagents. The work described herein can also be used to develop
model systems for the study of activation of mast cells through the
Fc.epsilon.RI receptor and to improve the therapeutic capability to
modulate the function of these cells.
[0048] Agents described herein can be anything which binds to or
interacts with CD81 and induces (i.e., activates) or enhances
CD81-mediated signal transduction. For example, the agent can be a
small molecule, a peptide, or a polyclonal or monoclonal antibody,
such as an anti-CD81 antibody. In particular embodiments, the
antibody is 5D1 or 1A12.
[0049] In order to identify membrane proteins capable of regulating
Fc.epsilon.RI signaling, mAbs to the rat basophilic leukemia
(RBL-2H3) cell line were produced and antibodies which inhibited
Fc.epsilon.RI-mediated degranulation were identified. The results
are shown in FIGS. 7A-7D. Cells were preincubated with mAb 5D1 or
buffer for 30 minutes (FIGS. 7A, 7B, 7D) or for the indicated times
(FIG. 7C) at room temperature prior to triggering for 30 minutes
(FIGS. 7A-7C) or as indicated (FIG. 7D). The data shown are
representative of more than 10 experiments with the 5D1 mAb. As
shown in FIG. 7A, pretreatment of anti-DNP IgE-saturated RBL-2H3
cells with purified mab 5D1 inhibited Fc.epsilon.RI-mediated
degranulation by 75%, as measured by release of granule-stored
.sup.3H-serotonin. Blockage of serotonin release was significant
(*, p<0.05) even at subsaturating concentrations of 5D1 (2.5 nm
mAb/10.sup.5 cells, FIG. 7A). 5D1-mediated inhibition was specific
for Fc.epsilon.RI signaling, as degranulation induced by phorbol
myristate acetate (PMA) and calcium ionophore ionomycin were
unaffected (FIG. 7B). Furthermore, maximal inhibition of
Fc.epsilon.RI-mediated degranulation by mAb 5D1 required only brief
periods of preincubation (FIG. 7C), and inhibition was sustained
for at least one hour of antigen stimulation (FIG. 7D).
[0050] The protein recognized by the degranulation-inhibitory 5D1
mAb was then identified. 5D1 and a second degranulation-inhibitory
mAb (1A12) recognized proteins of Mr 25 kDa. 5D1 and 1A12 blocked
each others'binding to RBL-2H3 cells, although neither mAb
inhibited IgE binding and, conversely saturation of Fc.epsilon.RI
with IgE had no effect on 1A12 binding, suggesting that 1A12 and
5D1 recognized the same protein (see FIG. 8) and that FC.epsilon.RI
and the lA12/5D1 antigen were not co-localized on the cell
membrane. Since mAb lA12 was more effective at immunoprecipitation
and on Western blots, it was used for protein purification. Batch
preparations of RBL-2H3 extracts were immunoprecipitated with mAb
1A12, resolved on preparative SDS-PAGE and transferred to
nitrocellulose for protein sequencing. Peptide sequence obtained
from Lys-C digests of lA12 immunoprecipitates is shown aligned with
homologous sequences from mouse and human CD81in FIG. 5. Based on
these data, rat CD81 was cloned from a RBL-2H3 cDNA library using
mouse CD81 cDNA as a probe and expressed in the mouse mast cell
line Cl.MC/C57.1 (Young et al., Proc. Natl. Acad, Sci. USA
84:9175-9179 (1987)). FACS profiles of Cl.MC/C57.1 transfectants
are shown in FIG. 8; both degranulation-inhibitory mAbs lA12 and
5D1 recognized rat CD81.
[0051] To target the site of CD81 inhibition of degranulation, the
effect of CD81 antibodies on the earliest events of Fc.epsilon.RI
signal transduction, i.e. tyrosine phosphorylation of proteins by
activated, nonreceptor tyrosine kinases including Lyn and Syk, and
calcium mobilization (Jouvin et al. , J. Biol. Chem. 269:5918-5925
(1994); Penhallow et al., J. Biol. Chem. 270:23362-23365 (1995);
Scharenberg et al., EMBO J. 14:3385-3394 (1995); Lin et al., Cell
85:985-995 (1996)) was examined. In these experiments,
IgE-saturated RBL-2H3 cells were pretreated with purified anti-CD81
prior to triggering with DNP-HSA for the indicated periods of time,
followed by extraction and immunoprecipitation of total
tyrosine-phosphorylated proteins. No major changes in the pattern
of Fc.epsilon.RI-induced tyrosine phosphorylation were detected
with anti-CD81 treatment prior to antigen triggering. Incubation of
RBL-2H3 cells with 5D1 alone (no antigen triggering) did not induce
detectable tyrosine phosphorylation.
[0052] The effect of anti-CD81 on Fc.epsilon.RI-induced calcium
mobilization was monitored on individual, adherent RBL-2H3 cells by
confocal microscopy in cells loaded with calcium dye fluo-3. As
shown in FIG. 9A, no inhibition of Fc.epsilon.RI-induced calcium
mobilization in anti-CD81 treated versus controls was observed by
confocal microscopy, despite inhibition of degranulation under
these conditions (FIG. 9B). Anti-CD81pretreatment had no effect on
calcium release from intracellular stores in cells triggered in
Ca.sup.2+-free buffer containing 0.5 mM EGTA or on pre-triggering
baseline values. Similar results were also obtained with RBL-2H3
triggered through Fc.epsilon.RI in suspension using a
spectrophotometer. In separate experiments, anti-CD81 mAb 5D1 did
not inhibit leukotriene C.sub.4 (LTC.sub.4) production induced by
DNP-HSA/IgE stimulation (FIG. 9C). LTC4 production is dependent on
activation of phospholipase A2 (tyrosine kinase and
calcium-dependent) and is regulated by PMA-sensitive, protein
kinase C isozymes (Currie et al., Biochem. J. 304:923-928 (1994));
Ali et al. , J. Immunol. 153:776-788 (1994)). These data suggest
that CD81 acts independently of early tyrosine phosphorylation and
calcium mobilization events which are critical for mast cell
degranulation.
[0053] These results were unexpected in light of the reported modes
of action of other inhibitory receptors. These proteins fall into
two major classes; type I, transmembrane proteins that are members
of the Ig superfamily (Fc.gamma.RIIb1 , KIR, CTLA-4, CD22, gp49bl,
paired Ig-like receptors (PIR), signal-regulatory proteins (SIRPs))
and type II, transmembrane, C-type lectins (e.g. Ly-49, NKG2A, mast
cell function associated protein (MAFA)) (Ono et al., Nature
383:263-266 1996); Burshtyn et al., Immunity 4:77-85 (1996)).
[0054] CD81 differs from these inhibitory receptors in three
important ways. First, unlike other inhibitory receptors, CD81
inhibits Fc.epsilon.RI-mediated degranulation while leaving both
tyrosine phosphorylation and calcium mobilization apparently
unaffected. While these results cannot exclude a very selective
inhibition of kinase activity by CD81 antibodies, it is clear that
no detectable effect is found on tyrosine kinase-sensitive calcium
mobilization of LTC.sub.4 production. Second, CD81 belongs to a
different structural class of proteins than the other inhibitory
receptors. CD81 is a TM4SF protein with four transmembrane spanning
segments, two extracellular loops, two short cytoplasmic tails, and
a short intracellular loop between transmembrane segments 2 and 3
(Wright and Tomlinson, Immuriol. Today 15:588-594 (1994)). Third,
the cytoplasmic tails of CD81 lack ITIM motifs. While there is an
ITIM-like sequence (GCYGAI) in the short intracellular loop between
transmembrane segments 2 and 3, there is no evidence that this site
is phosphorylated by tyrosine kinases or capable of binding to SH2
domains.
[0055] In order to assess the activity of anti-CD81 in
Fc.epsilon.RI signaling in normal mast cells, the passive cutaneous
anaphylaxis (PCA) model, a classic system for studying mast cell
activation in vivo (Wershil et al., J. Immunol. 154:1391-1398
(1995); Dombrowicz et al., J. Clin. Invest. 99:915-925 (1997)), was
chosen. In these experiments, rats were injected intradermally with
IgE mixed with anti-CD81 mAb 5D1 (IgGl) or with class-matched mouse
(IgG1) as control (FIG. l0A). Additional rats received anti-DNP IgE
alone into the skin at time 0, followed by a second injection
(buffer, 5D1, or anti-rat LFA-1.beta.(IgG1)) (FIG. l0B) into
IgE-injected sites 21 hours after IgE injections. Twenty four hours
after IgE priming, rats received 1 mg of antigen intravenously
(DNP-HSA containing 1% Evan's blue dye). Mast cell activation
through Fc.epsilon.RI in PCA results in the release of several
vasoactive substances which act to increase vascular permeability,
a property which is quantified by local accumulation of the Evan's
blue dye from the vasculature into the sites of IgE injections.
These results are expressed as .mu.g Evan's blue converted from
A.sub.610 measurements of formamide-extracted tissue biopsies
(Dombrowicz et al., J. Clin. Invest. 99:915-925 (1997)). As shown
in FIG. 10A, coinjection of anti-CD81 mAb 5D1 during IgE priming
significantly inhibited IgE-dependent PCA reactions (p=0.024)
compared to class-matched controls.
[0056] To limit the possibility of non-specific suppression of PCA
reactions due to tissue deposition of IgG.sub.1 mabs, these
experiments were repeated by injecting anti-CD81 mAb 5D1 or
anti-LFA-1.beta.(CD18) into the IgE-injected sites 3 hours before
antigen administration. LFA-1.beta.is expressed on mast cell lines
including RBL-2H3 but anti-LFA-1.beta.has no effect on
Fc.epsilon.RI-mediated degranulation in RBL,-2H3 cells (Weber et
al., Scand. J.Immunol. 45:471-481 (1997)). Similar to coinjection
of IgE and IgG.sub.1 mAbs, separate injections of anti-CD81 yielded
significant inhibition of PCA reactions compared to
anti-LFA-1.beta.controls (FIG. 10B).
[0057] Thus, it is demonstrated herein that CD81 is a novel
inhibitory receptor for Fc.epsilon.RI. The observation that CD81
acts on calcium-independent events required for mast cell
degranulation distinguishes CD81 from previously described
inhibitory receptors, such as Fc.gamma.TRIIb1 and KIR, which act
upstream of calcium influx. Anti-CD81 mAbs also inhibited
IgE-dependent PCA reactions, which suggests the CD81 pathway is
present in normal mast cells and capable of being engaged to
inhibit mast cell responses in vivo. Therefore, the CD81 inhibitory
pathway can be utilized in therapeutic strategies aimed at
intervention of allergic responses.
[0058] RBL-2H3 cells express Fc.epsilon.RI, CD81 and endogenous rat
Fc.gamma.RIII receptors. However, no high-affinity reagent
(antibody) is available to trigger the Fc.gamma.RIII receptors on
RBL-2H3; the 2.4G2 antibody (anti-mouse Fc.gamma.RII/Fc.gamma.RIII)
was used for this purpose. To demonstrate that CD81 stimulation
inhibits degranulation induced through Fc.gamma.RIII signaling as
it does for Fc.epsilon.RI, murine Fc.gamma.RIII.alpha. chain cDNA
was expressed in RBL-2H3 cells.
[0059] Fc.gamma.TRIII binding of IgG is detectable only when IgG is
present in the form of IgG-containing immune complexes which
crosslink Fc.gamma.TRIII receptors and initiate intracellular
signals. One of the methods of triggering Fc.gamma.RIII is through
stimulation with crosslinked anti-Fc.gamma.RIII antibodies. FIG. 12
shows the results when RBL-2H3 and Fc.gamma.RIII-transfectants of
RBL-2H3 were loaded with .sup.3H-serotonin in the presence (DNP-HSA
stimulation) or absence (immune complex stimulation) of
DNP-specific IgE. After overnight incubation, cells were washed and
incubated with culture media or media containing 200 ng of anti-rat
CD81 mAb 5D1 prior to triggering with optimized concentrations of
DNP-HSA or with preformed immune complexes of 2.4G2/anti-rat IgG
F(ab').sub.2. Degranulation was allowed to proceed for 30 minutes
at 37.degree. C. and released .sup.3H-serotonin was quantitated by
scintillation counting. As shown in FIG. 12, DNP-HSA induces
IgE-mediated degranulation in all four cell lines which is
inhibitable by anti-CD81 mAb 5D1. 2.4G2anti-rat IgG F(ab+)2
preformed complexes, but not anti-rat IgG F(ab)2 alone, induce
degranulation only in cells expressing mFc.gamma.RIII receptors
(RBL-2H3 transfectants A10, D10 and H11), a process which is also
inhibitable by preincubation with 5D1. This data provides the
identification of CD81 as a common inhibitor of both Fc.epsilon.RI
and Fc.gamma.RIII.
[0060] Accordingly, the present invention relates to a method of
inhibiting or enhancing cell surface receptor signaling, e.g.,
Fc.epsilon.RI-mediated or Fc.gamma.RIII-mediated signaling. The
method of inhibiting cell surface receptor signaling comprises
contacting a cell with an effective amount of an agent which
enhances or induces CD81-mediated signal transduction.
Alternatively, the method can be a method of inhibiting cell
surface receptor signaling in a mammal, comprising administering to
the mammal an effective amount of an agent which enhances or
induces CD81-mediated signal transduction. Appropriate cells are
any cell type which expresses or has been designed to express
(e.g., by transfection or genetic engineering) both CD81 and a
suitable cell surface receptor.
[0061] For example, inhibition of the cell surface receptor signals
which induce mast cell degranulation is useful in methods of
treating allergic conditions or inflammatory disorders. Enhancement
of the cell surface receptors which induce mast cell degranulation
is useful in inducing an inflammatory response, for example, in
response to bacterial or parasite infection.
[0062] The method of enhancing cell surface receptor signaling
comprises contacting a cell with an effective amount of an agent
which inhibits or prevents CD81-mediated signal transduction.
Alternatively, the method can be a method of enhancing cell surface
receptor signaling in a mammal, comprising administering to the
mammal an effective amount of an agent which inhibits or prevents,
CD81-mediated signal transduction. It may be clinically beneficial
to enhance cell surface receptor signaling in a mammal, or the
functional results thereof, such as degranulation, in conditions
where an inflammatory response and/or release of leukotrienes and
cytokines is beneficial, such as in host defense against parasites
and bacteria.
[0063] The invention also pertains to a method of treating an
allergy (e.g., asthma, hay fever or atopic eczema) or inflammatory
condition in a mammal comprising adminsitering to the mammal an
effective amount of an agent which induces CD81-mediated signal
transduction. For example, the method can be used to treat allergic
or inflammatory responses associated with disorders such as
autoimmune (Type I) diabetes mellitus, rheumatoid arthritis,
ankylosing spondylitis, sarcoidosis, Sjogren's syndrome, multiple
sclerosis, inflammatory bowel disease (i.e., Crohn's disease and
ulcerative colitis), dermatomyositis, scleroderma, polymyositis,
systemic lupus erythematosus, biliary cirrhosis, autoimmune
thyroiditis, and autoimmune hepatitis, as well as many
dermatological disorders, including psoriasis, contact sensitivity
and atopic dermatitis.
[0064] As used herein, "inhibit" is intended to encompass any
qualitative or quantitative reduction in a measured effect or
characteristic, including complete prevention, relative to a
control. As also used herein, "enhance" is intended to encompass
any qualitative or quantitative increase in a measured effect or
characteristic relative to a control. An "effective amount" of a
given agent is intended to mean an amount sufficient to achieve the
desired effect, e.g., the desired therapeutic effect, under the
conditions of administration, such as an amount sufficient for
inhibition or enhancement of CD81-mediated signal transduction.
[0065] The present invention also relates to preparations for use
in the inhibition or enhancement of cell surface receptor
signaling, and the treatment of allergic diseases and inflammatory
disorders, the preparation including an inhibitor or promoter of
CD81-mediated signal transduction, together with a physiologically
acceptable carrier and optionally other physiologically acceptable
adjuvants.
[0066] According to the method, a therapeutically effective amount
of one or more agents (e.g., a preparation comprising an inhibitor
or promoter of CD81-mediated signal transduction can be
administered to an individual by an appropriate route, either alone
or in combination with another drug.
[0067] A variety of routes of administration are possible
including, but not limited to, oral, dietary, topical, parenteral
(e.g., intravenous, intraarterial, intramuscular, subcutaneous
injection), and inhalation (e.g., intrabronchial, intranasal or
oral inhalation, intranasal drops) routes of administration,
depending on the agent and disease or condition to be treated. For
respiratory allergic diseases such as asthma, inhalation is a
preferred mode of administration.
[0068] Formulation of an agent to be administered will vary
according to the route of administration selected (e.g., solution,
emulsion, capsule). An appropriate composition comprising the agent
to be administered can be prepared in a physiologically acceptable
vehicle or carrier. For solutions or emulsions, suitable carriers
include, for example, aqueous or alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media.
Parenteral vehicles can include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's or fixed
oils, for instance. Intravenous vehicles can include various
additives, preservatives, or fluid, nutrient or electrolyte
replenishers and the like (See, generally, Remington's
Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., PA,
1985). For inhalation, the agent can be solubilized and loaded into
a suitable dispenser for administration (e.g., an atomizer,
nebulizer or pressurized aerosol dispenser).
[0069] Furthermore, where the agent is a protein or peptide, the
agent can be administered via in vivo expression of the recombinant
protein. In vivo expression can be accomplished via somatic cell
expression according to suitable methods (see, e.g. U.S. Patent No.
5,399,346). In this embodiment, nucleic acid encoding the protein
can be incorporated into a retroviral, adenoviral or other suitable
vector (preferably, a replication deficient infectious vector) for
delivery, or can be introduced into a transfected or transformed
host cell capable of expressing the protein for delivery. In the
latter embodiment, the cells can be implanted (alone or in a
barrier device), injected or otherwise introduced in an amount
effective to express the protein in a therapeutically effective
amount.
[0070] The invention also pertains to assays for identifying agents
which enhance or inhibit calcium independent CD81-mediated signal
transduction. The assay comprises combining a cell bearing CD81
with an agent to be tested, under conditions suitable for signal
transduction by CD81. The level or extent of CD81-mediated signal
transduction can be measured using standard methods and compared
with the level or extent of CD81-mediated signal transduction in
the absence of the agent (control). An increase in the level or
extent of CD81-mediated signal transduction relative to the control
indicates that the agent is a promoter of CD81-mediated signal
transduction; a decrease in the level or extent of CD81-mediated
signal transduction relative to the control indicates that the
agent is an inhibitor of CD81-mediated signal transduction.
[0071] Inhibitors or promoters of CD81-mediated signal
transduction, e.g., those identified by methods described herein,
can be assessed to determine their effect on cell surface receptor
signaling. Inhibitors or promoters of CD81-mediated regulation of
cell surface receptor signaling can be, for example, small
molecules, antibodies and/or peptides. A cell bearing CD81 and an
appropriate cell surface receptor (e.g., Fc.epsilon.RI or
Fc.gamma.RIII ) are combined with an inhibitor or promoter of
CD81-mediated signal transduction under conditions suitable for
signal transduction by both CD81 and the cell surface receptor. The
level or extent of cell surface receptor signaling can be measured
using standard methods and compared with the level or extent of
cell surface receptor signaling in the absence of the inhibitor or
promoter (control). An increase in the level or extent of cell
surface receptor signaling relative to the control indicates that
the agent is a promoter of cell surface receptor signaling; a
decrease in the level or extent of cell surface receptor signaling
relative to the control indicates that the agent is an inhibitor of
cell surface receptor signaling.
[0072] Cell surface receptor signaling can be measured directly,
such as by measuring the level or amount of an associated
signalling molecule, or indirectly, such as by a functional assay
measuring level or amount of degranulation or passive cutaneous
anaphylaxis.
[0073] The following Examples are offered for the purpose of
illustrating the present invention and are not to be construed to
limit the scope of this invention. The teachings of all references
cited herein are hereby incorporated herein by reference.
EXAMPLES
[0074] Cell Culture, Reagents and Antibodies The rat basophilic
leukemia cell line (RBL-2H3) was cultured in EMEM supplemented with
16% heat-inactivated FCS, 2 mM L-glutamine and penicillin (100
U/ml)/streptomycin (50 mg/ml) (Biofluids, Rockville, MD). NS-1 and
SP2/0 myeloma cells were cultured in RPMI 1640 supplemented with
20% FCS, glutamine and antibiotics. Cl.MC/C57.1 cells were cultured
as described in Young et al. (Proc. Natl. Acad. Sci. USA
84:9175-9179 (1987)). DNP-human serum albumin (DNP-HSA) (30-40
moles DNP/mole albumin) was purchased from Sigma Chemical Cc). (St.
Louis, MO). DNP-specific IgE supernatants were used to saturate
Fc.epsilon.RI as described in Young et al. (Proc. Natl. Acad. Sci.
USA 84:9175-9179 (1987)). For PCA experiments, MOPC31c (IgG.sub.1)
and anti-DNP-mouse IgE (clone SPE-7) were purchased from Sigma
Chemical Co. (St. Louis, MO) and anti-rat .beta.2 integrin
(anti-LFA-1.beta., CD18; clone WT.3) was purchased from Pharmigen
(San Diego, CA). MOPC 31c and anti-DNP IgE were dialyzed to remove
sodium azide before in vivo injections. Anti-rat CD81 (5D1,
IgG.sub.1) was purified from ascites on Protein G Sepharose
(Pharmacia, Uppsala, Sweden).
[0075] Immunizations, Fusions, and FACS Female BALB/c mice (4-8
weeks old) were immunized intraperitoneally with 25 x 10.sup.6
RBL-2H3 emulsified in complete Freund's adjuvant or 50
.times.10.sup.6 in PBS. Mice were boosted after 2 weeks with 40 x
10.sup.6 RBL-2H3 cells emulsified in incomplete Freund's adjuvant
intraperitoneally or in PBS. For the final immunizations, animals
were injected with 20-40 x 106 RBL-2H3 cells intraperitoneally at
day -4 (fusion =day 0) and intravenous at day -3. Spleen cell
preparations were fused with either NS-1 or SP2/0 myeloma cells in
polyethylene glycol and plated onto normal BALB/c spleen feeder
cells. To enhance the development of the hybridomas, S. typhimurium
mitogen (Ribi ImmunoChem Research, Inc., Namilton, MT) was included
in the culture medium from days 0-10. Hybridoma supernatants were
tested after day 14 by flow cytometry for binding to RBL-2H3 using
FITC-conjugated goat anti-mouse F(ab')2-specific antibody (Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA) and analyzed by
flow cytometry on a FACSCAN.TM. flow cytometer (Becton-Dickinson,
San Jose, CA).
[0076] From 3 separate fusions, a total of 2160 wells were plated
and 622 supernatants from wells with hybridoma growth were screened
by FACS for reactivity with RBL-2H3 cells. In all, 283/622 (45%)
elicited detectable reactivity by FACS with membrane antigens of
RBL-2H3. The screening of RBL-2H3-reactive mAbs by serotonin
release assay lead to the identification of 1A12 (IgG.sub.2b) and
5D1 (IgG.sub.1), which were characterized further. Rat CD81
transfectants of were stained with purified 1A12 and 5DI
(1.mu.g/10.sup.6 cells), counterstained with goat anti-mouse
F(ab').sub.2-specific antibody and analyzed by flow cytometry on a
FACScan.RTM. flow cytometer.
[0077] Serotonin Release Assay and Leukotriene C4 Assays
[0078] RBL-2H3 cells were loaded with .sup.[3H]5-hydroxytryptamine
.sup.[3H]serotonin; 0.1-0.3 mCi/10.sup.5 cells) and saturated with
DNP-specific IgE in 96-well microtiter tissue culture plates
(10.sup.5 cells/well, 37.degree. C., 5%- CO.sub.2) as described in
Da ron et al. (J. Immunol. 149:1365-1373 (1992)). Monolayers were
washed three times with buffer (glucose- saline, PIPES buffer (pH
7.2) containing (in mM) 25 PIPES, 110 NaCl, 5 KCl, 5.6 glucose, 0.4
MgCl.sub.2, l CaCl2 and 0.1% BSA), and 25 ml of a dilution of
purified antibody was added to the labeled monolayers, and plates
were incubated for 30 minutes (or as indicated) at room
temperature. Triggering of Fc.epsilon.RI was performed by the
addition of DNP-HSA (final concentration 10-250 ng/ml) and plates
were incubated at 37.degree. C. (except as indicated in FIG. 7D)
with control samples present on each plate. Degranulation was
stopped by placing the plates on ice and by the addition of 150
.mu.l of cold culture medium per well. 100 .mu.l aliquots were
taken from replicate wells for scintillation counting. Total
cellular incorporation was determined from 1% SDS/1% NP-40
lysates.
[0079] Leukotriene C4 was measured from 10.sup.6anti-DNP IgE
saturated RBL-2H3 treated with 1 .mu.g 5DI or buffer prior to
triggering with 30 ng/ml DNP-HSA. Supernatants were stored at
-80.degree. C. until measurement of LTC.sub.4 by specific enzyme
immunoassay (Cayman Chemical, Ann Arbor, MI).
[0080] Immunoaffinity Chromatography, Electrophoresis, and Western
Blotting
[0081] RBL-2H3 cells were cultured in routine culture medium in
spinner flasks to a cell density of approximately 10.sup.6/ml,
harvested by centrifugation and washed twice with cold PBS. Washed
cells were extracted in 0.5M K.sub.2HPO.sub.4 (pH 7.5) with
proteinase inhibitors (10 .mu.g/ml pepstatin, 5 .mu.g/ml leupeptin,
and 10 .mu.g/ml aprotinin) at 50.times.10.sup.6/ml for 60 minutes
at 4.degree. C. with frequent mixing. N-octyglucoside (10 mM) was
added during the extraction to ensure protein solubility.
Post-nuclear lysates were prepared by centrifugation at
15,000.times.g for 20 minutes at 4.degree. C. Lysates were then
passed through 0.2 mM filters to remove residual debris and passed
several times over protein G- Sepharose coupled to 1A12 (2 mg/ml
bed volume), washed with PBS (10 mM n-octylglucoside) and eluted
with 0.2 M glycine. Tris-neutralized, concentrated extracts were
reduced with .beta.-mercaptoethanol, resolved on 12.5% preparative
SDS-PAGE and transferred to Immobilon.sup.SQ (Millipore, Bedford,
MA). The membrane was stained with amido black and the Mr 25 kDa
band was excised, eluted, alkylated and digested overnight with
Lys-C. Peptides were separated by reverse phase-HPLC and the
peptide peak eluting at 36 minutes was sequenced. Subsequent
cloning and expression of rat CD81 cDNA from RBL-2H3 confirmed that
1A12 and 5D1 recognize rat CD81 and that CD81 crosslinking inhibits
Fc.epsilon.RI-mediated mast cell degranulation.
[0082] For anti-phosphotryosine Western blots, 0.5% Triton X-100
(BBS, proteinase inhibitors) extracts were immunoprecipitated
overnight with 2 .mu.g of anti-phosphotryosine mAb 4G10 bound to
protein A-Sepharose beads (4.degree. C with rotation). Beads were
washed with lysis buffer, eluted, resolved on 12.5% SDS-PAGE,
transferred to nitrocellulose membranes and immunoblotted with 1
.mu.g/ml 4G10 mAb, followed by incubation with HRP-conjugated
anti-mouse IgG secondary antibodies and development with
chemiluminescence substrates (Renaissance, Dupont/NEN, Boston, MA).
Construction and Screening of RBL-2H3 cDNA library in UNI-ZAP.TM..
Poly A+ mRNA was isolated from RBL-2H3, reverse-transcribed into
cDNA, size-fractionated on Sephacryl S-500 spin columns and ligated
into UNI-ZAP-XR lambda vector according to the manufacturer's
instructions (Stratagene, La Jolla, CA). After rescue of the cDNA
inserts and appropriate restriction enzyme digests, it was
determined that 96% of the plamids contained inserts, with an
average size of 1.7 kB. 5.times.10.sup.5 plaques were screened with
.sup.32P-labeled mouse CD81 cDNA probe. After hybridization,
nitrocellulose filters were washed once with 2.times.SSC containing
0.1% SDS (room temperature) and 3 times with 0.5x SSC containing
0.1% SDS at 50.degree. C. Filters were autoradiographed and plaques
picked and eluted. Candidate plaques were subjected to three
additional rounds of plaque purification before rescue of the cDNA
inserts into pBluescript. Sequencing was performed on eleven
isolates and all were found to align with accession number U19894
isolated from rat brain (Geisert, Jr., et al., Neurosci. Lett.
133:262-266 (1991); Irwin and Geisert, Jr., Neurosci. Lett.
154:57-60 (1993); Geisert, Jr., et al., J. Neurosci. 16:5478-5487
(1996)).
[0083] Transfections: Rat CD81 cDNA from two isolates was subcloned
into the pBJ1neo expression vector (Lin et al., Cell 85:985-995
(1996)) and 20 .mu.g of ethanol-precipitated DNA was used for
electroporation of C1.MC/C57.1 cells (1050 .mu.F, 270v). Selection
of stable transfectants was initiated 48 hours later by replating
at 500-10,000 cells per well with 2 mg/ml G418 (Life Technologies,
Grand Island, NY).
[0084] Confocal Microscopy: After overnight adherence and
saturation of Fc.epsilon.RI with DNP-specific IgE, RBL-2H3 cells
were washed with buffer and incubated with 3 .mu.M fluo3AM
(Molecular Probes, Eugene, OR) and 0.2 mg/ml Pluronic (Molecular
Probes) at 37.degree. C for 30 minutes (5% CO.sub.2) in a buffer
containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2,
10 mM glucose, and 1 mM Na-HEPES (pH 7.4). Dye-loaded cells were
then washed once with the same buffer before preincubation (30
minutes, room temperature) with buffer (.+-.5D1, 1
.mu.g/chamber/10.sup.5 cells) and triggering with 100 ng/ml
DNP-HSA. Ca.sup.2+measurements in single cells were monitored using
a laser-scanning confocal microscope (LSM4, Zeiss, New York, NY)
equipped with an argon/kryton laser to excite the dye at 488 nm.
Fluorescence emission above 510 nm was measured after placing a
long pass filter in front of the photomultiplier tube. The confocal
system was employed in slow scan mode and fluorescence images were
collected every 5 seconds. Fluo-3 fluorescence measurements were
normalized by dividing the average fluorescence intensity (F)
occurring during the course of the experiment to the average
fluorescence intensity determined at the beginning of the
experiment (F.sub.c). All measurements were performed at
22-24.degree. C.
[0085] Passive Cutaneous Anaphylaxis in Rats
[0086] Male Wistar rats (275-300 g) were used in these experiments.
Rats were first anesthetized with ether, then back skin hair was
shaved and rats were injected intradermally with 50 .mu.l
containing 100 ng anti-DNP IgE or 25 ng anti-DNP-IgE mixed with 50
.mu.g of MOPC 31c (mouse IgG.sub.1, specificity unknown) or 5D1
(mouse IgG.sub.1, anti-rat CD81). Control sites received buffer
alone (PBS containing 10 .mu.g/ml mouse serum albumin; Sigma
Chemical Co., St. Louis, MO). Sites were marked on the skin for
orientation and rats that received 100 ng anti-DNP injections
received a second injection 21 hours later with 50 .mu.g of 5D1 or
anti-rat LFA-1.beta.(CD18; mouse IgG.sub.1) into previously
injected sites. Sites receiving IgE and IgG.sub.1 were injected in
triplicate on the same rat. Twenty-four hours after IgE injections,
animals received 1 ml of 1 mg/ml DNP-HSA containing 1% Evan's Blue
dye injected intravenously under ether anesthesia. Thirty minutes
after intravenous injection, rats were sacrificed, and punch
biopsies (2.5 cm.sup.2) were obtained, minced and extracted 3 times
with hot formamide (80.degree. C, 3 hours) (Dombrowicz et al., J.
Clin. Invest. 99:915-925 (1997)). Pooled samples from tissue sites
were centrifuged and absorbance at 610 nm (A.sub.610) was measured.
A.sub.610 values were converted to .mu.g Evan's blue based on a
standard curve of dilutions of Evan's Blue in formamide.
[0087] Inhibition of Signaling Elicited Through the Low Affinity
IgG Receptor FcR.gamma.III
[0088] RBL-2H3 cells express Fc.epsilon.RI, CD81 and endogenous rat
Fc.gamma.RIII receptors. However, no high-affinity reagent
(antibody) is available to trigger these receptors on RBL- 2H3; the
2.4G2 antibody (anti-mouse Fc.gamma.RII/Fc.gamma.RIII) was used for
this purpose. To demonstrate that CD81 stimulation inhibits
degranulation induced through Fc.gamma.RIII signaling as it does
for Fc.epsilon.RI, murine Fc.gamma.RIII.alpha. chain cDNA was
expressed in RBL-2H3 cells. FcR.gamma. cDNA was cotransfected to
assist in the surface expression of Fc.gamma.RIII complexes. In
FIGS. 11A-11D, the histograms of 3 stable mouse Fc.gamma.RIII
RBL-2H3 transfectants are shown after staining with 2.4G2 and
FITC-anti-rat IgG. Untransfected RBL-2H3 cells exhibit no
detectable binding of 2.4G2 (FIG. 11A).
[0089] Fc.gamma.RIII binding of IgG is detectable only when IgG is
present in the form of IgG-containing immune complexes which
crosslink Fc.gamma.RIII receptors and initiate intracellular
signals. One of the methods of triggering Fc.gamma.RIII is through
stimulation with crosslinked anti-Fc.gamma.RIII antibodies. In FIG.
12, RBL-2H3 and Fc.gamma.RIII -transfectants of RBL-2H3 were loaded
with .sup.3H-serotonin in the presence (DNP-HSA stimulation) or
absence (immune complex stimulation) of DNP-specific IgE. After
overnight incubation, cells were washed and incubated with culture
media or media containing 200 ng of anti-rat CD81 mAb 5D1 prior to
triggering with optimized concentrations of DNP-HSA or with
preformed immune complexes of 2.4G2/anti-rat IgG F(ab')2.
Degranulation was allowed to proceed for 30 minutes at 37.degree. C
and released .sup.3H-serotonin was quantitated by scintillation
counting. As shown in FIG. 12, DNP-HSA induces IgE-mediated
degranulation in all four cell lines which is inhibitable by
anti-CD81 mAb 5D1. 2.4G2/anti-rat IgG F(ab')2 preformed complexes,
but not anti-rat IgG F(ab)2 alone, induce degranulation only in
cells expressing mFc.gamma.RIII receptors (RBL-2H3 transfectants
A10, D10 and H11), a process which is also inhibitable by
preincubation with 5D1. This data provides the identification of
CD81 as a common inhibitor of both Fc.epsilon.RI and
Fc.gamma.RIII.
Results
[0090] 5D1 mAb inhibits Fc.epsilon.RI-mediated degranulation by
antigen. From 3 separate fusions, a total of 2160 wells were plated
and 622 supernatants from wells with hybridoma growth were screened
by FACS for reactivity with the immunizing RBL-2H3 cells (see Table
1). In all, 283/622 elicited detectable reactivity by FACS with
membrane antigens of RBL-2H3. Supernatants from the positive
hybridomas were then tested for inhibition of
Fc.epsilon.RI-mediated degranulation. RBL-2H3 cells exhibit a
reproducible degranulation profile to Fc.epsilon.RI-IgE stimulation
by the corresponding antigen DNP-HSA. Detectable serotonin release
is observed with 1 ng/ml concentrations of DNP-HSA; maximal
serotonin release occurs with approximately 50 ng/ml, and at
concentrations greater than 1 mg/ml DNP-HSA degranulation is
inhibited, presumably because of the diminished ability of large
Fc.epsilon.RI-IgE aggregates to signal. In FIG. 4, purified 5D1 mAb
inhibits IgE-mediated degranulation in RBL-2H3 cells stimulated
with 10, 50 or 250 ng/ml DNP-HSA, with maximal inhibition occurring
at 5-20 ng/10.sup.5 RBL-2H3 cells. RBL-2H3 cells were saturated
with DNP-specific IgE and labeled with 3H-hydtroxytryptamine
(serotonin) 0.2 mCi/10.sup.5 cells/well (0.32 cm.sup.2), washed
three times with triggering buffer and incubated for 30 minutes at
room temperature with the indicated concentration of
affinity-purified 5D1 mAb in 25 ml total volume. After incubation,
cells were challenged with 25 .mu.l of 2X dilution of pre-warmed
DNP-HSA and triggered for 30 minutes (37.degree. C, 5% CO.sub.2).
Release was terminated by the addition of 150 .mu.l of ice-cold
triggering buffer and by placing the plates on ice. 100 .mu.l
aliquots of released radioactivity as well as SDS cell lysates were
then harvested and scintillation counted. Degranulation-inhibitory
mAb binding has little or no effect on IgE or
anti-Fc.epsilon.RI.alpha. binding.
1TABLE 1 Binding inhibition of FITC-conjugated mAbs directed to
RBL-2H3 surface antigens Median Fluorescence Intensity (MFI)
FITC-conjugated mAbs Preincubation Specificity 1A12 IgE 4H7 3A9 --
-- 37.9 75.0 83.5 289.0 1A12 ND 61.0 83.5 289.0 5D1 6.5 ND ND ND
4H7 rat Fc.epsilon.RI.alpha. 38.2 6.4 7.2 38.5 3A9 rat
Fc.epsilon.RI.alpha. 37.5 6.5 6.8 13.4 BC4 rat Fc.epsilon.RI.alpha.
38.5 5.8 5.4 5.9 5.14 rat Fc.epsilon.RI.alpha. 39.6 5.5 84.3 161.0
AA4 ganglioside ND 12.3 59.3 201.7 G.sub.dlb
[0091] 10.sup.6 RBL-2H3 cells were incubated on ice with a
saturating amount of unconjugated antibody (preincubation) for 30
minutes prior to the addition (without washes) of a titered
(subsaturating) concentration of FITC-conjugated mAb. After washes,
stained cells were analyzed by FACS and mean values histgram peaks
converted to median fluorescent intensity (MFI) units.
Equivalents
[0092] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the following
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
* * * * *