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Clinical and Diagnostic Laboratory Immunology, November 1999, p. 856-860, Vol. 6, No. 6
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of a Subset of Common Variable Immunodeficiency
Patients with Impaired B-Cell Protein Tyrosine
Phosphorylation
Rivka
Schwartz,1
Yael Ben-Anat
Porat,1
Zeev
Handzel,2
Zeev
Sthoeger,2
Ben-Zion
Garty,3
Ronit
Confino-Cohen,4
Jacov
Levy,5 and
Israel
Zan-Bar1,*
Sackler Faculty of Medicine, Tel-Aviv
University, Tel Aviv,1 Kaplan Hospital,
Rehovot,2 Schneider Children's
Hospital, Petach Tikva,3 Meir Hospital,
Kfar Saba,4 and Soroka Medical Center,
Beer Sheva,5 Israel
Received 29 January 1999/Returned for modification 25 March
1999/Accepted 18 August 1999
 |
ABSTRACT |
The mechanisms responsible for common variable immunodeficiency
syndrome (CVID) are as yet unknown. In the present study, we show that
the B-cell dysfunction in a subset of CVID patients is caused by
defective protein tyrosine phosphorylation (PTP). We demonstrated that
the PTP level and immunoglobulin (Ig) secretion malfunctions can be
successfully repaired when normal plasma membrane components are
implanted into these patients' B cells. Stimulation of CVID patients'
peripheral blood mononucleated cells with anti-Ig antibody revealed
that 7 of 11 patients had lower PTP levels than those found in the
normal donor cells. Plasma membrane implantation to the cells of these
patients resulted in elevated PTP levels which reached normal levels
upon stimulation with anti-human Ig antibody. The results revealed two
distinct groups of CVID patients. The first group included patients
whose B cells expressed low PTP levels after Ig stimulation. In these
patients the plasma membrane implantation restored the normal PTP level
as well as the ability to secrete IgM and/or IgG after B-cell
stimulation. In the second group, patients whose B cells expressed a
normal PTP level after Ig stimulation, with no restoration of their
ability to secrete Ig upon plasma membrane implantation and
lipopolysaccharide stimulation. We conclude that the first group has an
early signal transduction defect located in the B-cell plasma membrane,
while in the second group the defect is located elsewhere.
| |
INTRODUCTION |
Common variable immunodeficiency
syndrome (CVID) is a heterogeneous group of disorders characterized by
hypogammaglobulinemia and recurrent bacterial infections, particularly
involving the gut and the upper and lower respiratory tracts, which are
a direct result of deficiency in antibody production (6, 26,
30).
Most CVID patients have normal numbers of mature B cells in the
peripheral blood and lymphoid tissues. However, their B cells are
unable to differentiate normally into immunoglobulin (Ig)-secreting plasma cells (26, 30). Thus far, the primary immunologic
cause(s) responsible for this defect in B-cell differentiation is not known.
Initial studies on CVID syndrome found that peripheral blood
mononuclear cells (PBMCs) from CVID patients were unable to secrete normal amounts of Igs when stimulated in vitro with the lectin pokeweed
mitogen (5, 29). In a later study, patients with CVID were
classified on the basis of the ability of their B cells to secrete IgM
and/or IgG in response to interleukin-2 (IL-2) and anti-Ig antibody
stimulation in vitro (4, 7, 10, 11). In addition to B-cell
abnormalities, a variety of T-cell functional defects have been
described in many patients. These functional defects consist of reduced
proliferation rate and IL-2 secretion in response to various T-cell
stimuli (24, 27, 28) and an excessive suppressor T-cell
function (2, 26).
In the present study, we investigated whether the B-cell defect which
may cause this syndrome is related to the membranal receptors or
membranal enzymes which participate in signal transduction cascade. We
show that the functional defects are caused by defective tyrosine
phosphorylation in B-cell signal transduction of a subset of CVID
patients. We used plasma membrane (PM) implantation, which provides the
patients' B cells with normal receptors and membranal enzymes
(18), in an attempt to restore the protein tyrosine phosphorylation (PTP) level and to repair the Ig secretion malfunctions.
| |
MATERIALS AND METHODS |
Patients.
PBMCs were taken from 13 nonrelated CVID patients,
eight adults (two males and six females; age range, 20 to 66 years) and five children (two males and three females; age range, 1.5 to 10 years), and 29 normal donors. The serum Ig levels in the adults were
<219 mg of IgG, <30 mg of IgM, and <10 mg of IgA per dl and in the
children were <50 mg of IgG, <15 mg of IgM, and <8 mg of IgA per dl.
Nonetheless, upon counting and staining of their PBMCs with anti-IgM,
-CD3, -CD4, -CD8, and -CD19 antibodies, all CVID patients were shown to
have normal quantities and phenotypes of B and T cells. Numbers of
PBMCs in adults ([1.3 to 2.1] × 106) and in children
([2.4 to 3.2] × 106) were as follows: Ig+
cells, 7 to 14%; CD19+ cells, 8 to 17%; CD3+
cells, 54 to 64%; CD4+ cells, 29 to 54%; CD8+
cells, 15 to 38% (lowest to highest values). All CVID patients had
been treated routinely with intravenous injections of Igs.
Cell separation.
PBMCs were obtained from heparinized venous
blood of CVID patients and age- and sex-matched normal donors. Each
patient was bled three to four times at intervals of 0.5 to 1 year. The
PBMCs were isolated by gradient centrifugation in Ficoll-Paque
(21) and incubated in culture medium containing RPMI 1640 supplemented with 0.01 M HEPES, 0.1 M NaHCO3, 2 mM
L-glutamine, 1 µg of kanamycin per ml, 100 U of
penicillin per ml, 100 U of neomycin per ml, 100 U of streptomycin per
ml, and 10% heat-inactivated fetal calf serum.
Measurements of PTP levels.
PBMCs (7.5 × 106) were cultured in 1 ml of RPMI 1640 in the presence of
20 µg of goat anti-human IgM f(ab')2 antibody (Jackson Immunoresearch Laboratories ICN, West Grove, Pa.) for various times for
up to 30 min at 37°C (maximal response was observed after 10 min).
The cells were lysed by the addition of 1 ml of lysis buffer (20 mM
Tris-HCl, pH 8; 137 mM NaCl; 10% glycerol; 2 mM EDTA; 1 mM
NaVO4; 1% Triton X-100; 1 mM phenylmethylsulfonyl fluoride; 20 µM leupeptin; 0.15 U of aprotinin per ml) and incubated for 45 min at 4°C. Lysates were clarified by centrifugation at 6,000 rpm for 15 min, and the supernatants were collected. The cell lysates
(60 µg/lane) were separated on 10% polyacrylamide gels and
transferred to nitrocellulose membrane. Tyrosine phosphorylation was
detected by immunoblotting with biotinylated antiphosphotyrosine monoclonal antibody (BioMaker; Kiriat Weizmann, Rehovot, Israel), horseradish peroxidase-conjugated streptavidin (Amersham International, Plc., Little Chalfont, Buckinghamshire, United Kingdom), and
enzyme-linked chemiluminescence (ECL) (16). Levels of PTP
were determined by densitometric analysis and are expressed as a
densitometry unit (optical density times the area).
Ig secretion response.
Cells (0.5 × 106)
were cultured in flat-bottom microtiter plates with 2 µg of LPS per
ml for 7 days at 37°C in humidified 5% CO2. Cell culture
supernatants were analyzed for IgM, IgG, and IgA content by
enzyme-linked immunosorbent assay (ELISA) by using biotin-conjugated
goat anti-human IgM, IgG, or IgA antibodies, streptavidin alkaline
phosphatase (Amersham International Plc.), and phosphatase substrate
(p-nitrophenylphosphate disodium) (Sigma Chemical Co., St.
Louis, Mo.) (25). In order to present the Ig quantities in
all experiments performed, we calculated the stimulation index in each
experiment by dividing the quantity of Ig secreted in LPS-stimulated
cells by that secreted in nonstimulated cells. The isotype levels in
the nonstimulated cultures ranged from 150 to 250 ng of IgM, 50 to 150 ng of IgG, and 10 to 45 ng of IgA per ml and in the stimulated cultures
were up to 1,000 ng of IgM, 500 ng of IgG, and 90 ng of IgA per ml.
PM implantation.
The implantation of the foreign PM was
carried out by fusion of normal functional murine lymphocyte PM
vesicles to the patients' PBMCs by intact noninfectious Sendai virus
(SV) (1, 17, 18, 22).
PM vesicles were prepared from murine spleen cells as previously
described (1, 18). Briefly, murine spleen cells were suspended in hypotonic solution and were disrupted by use of a glass
Teflon Potter homogenizer and a rotor-driven pestle. The disrupted cell
suspension was centrifuged on 22% sucrose cushion for 60 min at
100,000 × g, and the interface suspension was
collected and stored at 
70°C.
Fusion of the PM vesicles to the PBMCs was carried out in a two-step
procedure (1, 17, 22). First, 0.06 µg of SV was incubated
with 4 µg of PM vesicles in a total volume of 100 µl of
phosphate-buffered saline for 10 min in 4°C and then for 30 min in
37°C to allow fusion of the virus to the PM. Second, the PM-SV
vesicles were fused to PBMCs under the same conditions as in the first
step (18). Immediately after implantation, cells were
stimulated, and Ig secretion and the PTP rate were measured as
described above. The above quantities, as well as the ratio between
viral units and PM, were optimal for maximal PM implantation per cell
(18). Implantation of the SV moiety alone was shown before
to have no effect on murine and human lymphocyte activation, proliferation, or Ig secretion (3, 23).
Surface staining of implanted normal cells with FITC-labeled
anti-mouse Ig antibody.
Normal PBMCs were implanted with murine PM
vesicles, stained with fluorescein isothiocyanate (FITC)-conjugated
anti-mouse Ig antibody. The stained cells were analyzed by use of a
flow cytometer apparatus (FACStar; Becton Dickinson, San Jose, Calif.).
Statistical analysis.
Analysis of variance was used to
determine significance.
| |
RESULTS |
PTP level in CVID B cells after stimulation through B-cell receptor
(BCR).
In an initial set of experiments, we tested the hypothesis
that CVID syndrome is related to defects in the early signal
transduction events.
We therefore examined the phosphotyrosine kinase function by measuring
the PTP level in CVID and normal donor B cells after stimulation with
anti-human Ig antibody. Since the kinetics in protein tyrosine
phosphorylation were found to be the same in both CVIDs and normal
controls, all of the results presented are at 10 min of stimulation,
which was found to be the peak response time.
The Western blotting figure of two representative CVID patients and
normal control PTP level studies shown in Fig.
1 were from one patient with low and one
patient with normal PTP levels. The complete results of these studies
in eleven patients are given in densitometry units in Table
1.
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FIG. 1.
PTP levels of representative CVID PBMCs stimulated with
anti-human IgM antibodies. PBMCs (7.5 × 106) of CVID
patients (P) and normal controls (N) were stimulated with goat
anti-human IgM f(ab')2 antibodies (20 µg/ml) for 10 min
and lysed in a lysis buffer. PTP levels of cell lysates were visualized
by Western blot with antiphosphotyrosine antibodies and ECL.
|
|
The results demonstrate that B cells of 7 of 11 CVID patients express
low PTP levels compared to normal (reduction of more than 30%) after
stimulation through BCR, suggesting an early signal transduction defect
in B cells in more than one-half of the patients. The maximum variation
in the PTP levels of all the examined normal donors was lower than
13%. This value was obtained in an experiment in which the PTP levels
were measured in PBMCs of 11 normal donors and were run simultaneously
on the same gel. The mean ± the standard deviation (SD) of all
densitometry units measurements was 1 ± 0.13. Therefore,
measurements that exceeded two SD from the mean (>0.74) indicated a
significant reduction in enzyme activity.
PTP levels in implanted CVID B cells after stimulation through
BCR.
We next tested the possibility that the signal transduction
defect in CVID B cells is related to membranal receptors or enzymes which participate in the signal transduction cascade. This was tested
by implanting normal murine PM in CVID PBMCs.
First, we confirmed the success of PM implantation by demonstrating the
existence of the foreign murine PM in the normal implanted cell
membranes. As shown in Fig. 2, all of the
implanted cells contained the murine PM in their cell membrane. The
PBMCs of five CVID patients whose B cells had a low PTP level after
stimulation through BCR, as well as of normal donors, were implanted
with normal murine PM. After verification of implantation in all PBMCs, the implanted cells were stimulated with anti-human Ig antibodies, and
their PTP levels were measured. The Western blotting figure of PTP
levels in one representative CVID and one normal control is shown in
Fig. 3, and the complete results of these
measurements carried out in the five patients and five controls are
given in densitometry units in Table 2.
As shown, almost normal PTP levels were achieved after PM implantation
and BCR stimulation in PBMCs of those patients whose levels had been
low. These results indicate that B cells of these patients exhibit an
early signal transduction defect which is located in their plasma
membrane. This defect can be repaired by implantation of normal PM.
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FIG. 2.
Flow cytometric analysis of PM implanted cells. PBMCs
(106) were implanted as described in Material and Methods
with PM originated from murine splenic cells. Implanted (in white) and
nonimplanted (in black) PBMCs were stained with FITC-conjugated
anti-mouse Ig antibody and analyzed by flow cytometry.
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|
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FIG. 3.
PTP levels of representative PM implanted CVID PBMCs
stimulated with anti-human IgM antibodies. PBMCs of a CVID patient (P1)
and a normal donor (N) were implanted with functional PM and stimulated
with anti-human IgM, and PTP levels were measured as described in Fig.
1.
|
|
Ig responses of CVID B cells to LPS.
In this set of
experiments, we examined whether the implantation of functional PM in
CVID PBMCs can not only restore the earliest events of the signal
transduction cascade through their BCR but also restore their ability
to secrete Ig in vitro in response to LPS stimulation. Implanted and
nonimplanted CVID and normal donor PBMCs were stimulated with LPS, and
their Ig isotype secretion was assessed.
As shown in Fig. 4, upon stimulation with
LPS, implanted lymphocytes of 5 of 9 patients exhibited significantly
elevated IgM secretion; two of the five also exhibited IgG secretion,
while none of the implanted patients exhibited restoration of IgA
secretion.
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FIG. 4.
In vitro Ig secretion of CVID implanted cells. CVID
patients (P1 to P9) and normal control (N) PBMCs were implanted with
functional PM as described in Materials and Methods. A total of 5 × 105 implanted and nonimplanted PBMCs were cultured with
2 µg of LPS per ml. Ig titers in 7-day cell culture supernatants were
determined by isotype-specific ELISA. The data are presented as the
mean ± the SD of the stimulation index from three experiments in
each patient, each of which was performed in triplicate. N represents
the mean of 14 normal controls. An asterisk indicates statistical
significance.
|
|
Our overall findings suggest that a membranal defect may be the cause
of B-cell dysfunction in a subset of CVID patients.
| |
DISCUSSION |
Most patients with CVID have normal numbers of mature B cells in
the peripheral blood and lymphoid tissues, but these cells fail to
proliferate and/or differentiate into Ig-secreting cells when
stimulated with mitogens (26). The study reported here demonstrates that an abnormality exists in the early signal
transduction cascade of B cells in a significant group of CVID patients.
We found that, after anti-IgM stimulation, the PTP levels in B cells of
more than one-half (7 of 11) of the patients were significantly low
compared to normal donors, suggesting abnormality in quantity and/or
activity of either PTKases or PTPases located in the plasma membranes
of these patients. Implantation of functional PM in the B cells of
these patients restored their levels to normal.
It is unclear whether the PM implantation provided a substitute for the
receptors or for the membrane-associated enzymes that may be defective
in CVID patients' B cells. However, since the stimulation of the
implanted B cells was carried out by anti-human Ig antibody and since
the cells were implanted with murine PM, it appears that the membranal
defective components in CVID B cells are the membrane-associated
enzymes and that the sIg receptors are unimpaired. Moreover, this leads
to the assumption that the functional improvement of the implanted
cells results from the activity of the murine membranal enzymes.
Additional studies to rigorously address these possibilities will be required.
In the PM implantation system one has to take in account that we are
using the PM of murine splenic cells which is implanted in both the B
and T cells of the PBMCs. However, as the cells are stimulated with
anti-human Ig antibodies which do not cross-react with the murine
surface Ig, we are thus stimulating only the implanted human B cells.
The results of this study are consistent with those of previous studies
demonstrating defects in the signal transduction cascade of B cells of
CVID patients: low or no elevation of free intracellular calcium
concentration and low expression of c-myc mRNA upon anti-Ig stimulation
of the cells (19, 20). Furthermore, similar data on the T
cells of CVID patients were obtained by Fischer et al. (13-15), who found a defect in the elevation of free
intracellular calcium in response to superantigen in T cells of some
CVID patients, whereas the response to phorbol myristate acetate and
ionomycin, which bypass receptor-mediated signaling, was unimpaired.
Next, we investigated whether the low PTP levels after stimulation
through BCR may be the cause of hypogammaglobulinemia in CVID patients.
To this end, we tested whether the implantation of functional PM into
the patients' PBMCs can restore, in addition to PTP levels, the
ability to secrete IgM, IgG, and IgA in response to LPS.
The results demonstrate that in a group of CVID patients the Ig
secretion defect can be at least partially restored when B cells are
implanted with functional PM and stimulated with LPS. These findings
are consistent with previous findings from our laboratory demonstrating
repair in the secretion of serotonin by murine implanted mast cells
(1).
In the present study we have noticed two distinct groups of CVID
patients with regard to the ability of PM implantation to restore their
B-cell defect(s). The first group included two patients (P1 and P5)
expressing low PTP levels in whom PM implantation restored both their
PTP levels and their ability to secrete IgM after B-cell stimulation.
We suggest that in this group of patients the main defect is an early
signal transduction defect which is located in their B-cell plasma
membrane. The second group included three patients (P3, P6, and P7)
expressing normal levels of PTP in whom PM implantation did not restore
their ability to secrete Ig upon LPS stimulation. We suggest that in
this group of patients the defect is not located in their B-cell plasma
membrane but may be located downstream in their signal transduction pathway.
It seems that the B cells of patient 2 (P2) possess at least two
defects in their signal transduction cascade: one located in the plasma
membrane and the other located in the cytoplasm or nucleus. This
patient's B cells express low PTP levels that can be restored by PM
implantation, with no repair of their Ig secretion upon B-cell
stimulation. A possible explanation is that in this patient's B cells
there is more than one defect, one related to receptor-associated
enzymes located in the plasma membrane and another located downstream
in the signal transduction pathway which therefore cannot be restored
by PM implantation.
It is noteworthy that in response to LPS, implanted lymphocytes of 5 of
9 patients significantly elevated IgM levels and two of the five also
exhibited IgG secretion, while none of the implanted patients exhibited
restoration of IgA secretion. This partial restoration of Ig secretion
may indicate an additional defect in the cells' isotype-switching
mechanism which cannot be restored by PM implantation. A possible
explanation for the cause of such a defect is a block in CD40-CD40
ligand (8). The CD40-CD40 ligand signal interaction defect
can be either common to or different from the BCR signal-transduction
defect. This explanation is supported by previous studies which
demonstrated a defect in CD40 ligand expression by activated
lymphocytes from a group of CVID patients (9, 12). However,
further studies are needed to examine this possibility.
In summary, the findings presented here suggest an early signal
transduction defect as the cause for the block in B-cell
differentiation in a group of CVID patients. Further studies are now
being carried out to determine the specific molecular defect in the
signal transduction pathway and the role of the receptor itself in
these cells.
| |
ACKNOWLEDGMENTS |
This work was supported in part through grants from the Israel
Academy of Science and Humanity and the Israel Research Fund. This work
was done in partial fulfillment of the requirements for the Ph.D.
degree of Rivka Schwartz from the Sackler Faculty of Medicine, Tel Aviv University.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978 Israel. Phone: 972-3-640-9920. Fax: 972-3-640-9160. E-mail: zanbar{at}post.tau.ac.il.
| |
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Clinical and Diagnostic Laboratory Immunology, November 1999, p. 856-860, Vol. 6, No. 6
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
