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*
Institute for Advanced Medical Research and
Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan; and
Department of Internal Medicine, Tokyo Electric Power Company Hospital, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The spleen is thought to play an important role in the pathogenesis of
IMTP, because 
60% of IMTP patients achieve a stable increased
platelet count after surgical splenectomy (7, 8). In
chronic IMTP, the spleen is considered to be the primary site of both
platelet destruction and anti-platelet Ab production
(7). Destruction of the Ab-sensitized platelets by
phagocytosis through FcR in the reticuloendothelial system, including
the spleen, has been confirmed by the clinical benefit of anti-Fc
blockade treatment using anti-Rho(D) Ig (9) or Abs to
the FcR (10). In contrast, the evidence for
anti-platelet Ab production by spleens from IMTP patients was
previously shown by McMillan et al. (11) and Karpatkin et
al. (12). These studies demonstrated that splenocytes
from IMTP patients spontaneously produce IgG Abs capable of
binding specifically to autologous and allogeneic platelets. The daily
splenic production of anti-platelet IgG was shown to exceed the
quantity required for maximum sensitization of platelets produced daily
in the bone marrow (11). If this is the case, autoreactive
T and B cells responsive to gpIIb-IIIa should be activated and expanded
in the spleen rather than in the peripheral blood. To test this
hypothesis, the frequencies and activation status of
gpIIb-IIIa-reactive T and B cells were compared between PBMCs and
splenocytes obtained from IMTP patients who received surgical
splenectomy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nine adult patients with chronic IMTP who received surgical
splenectomy between July 1998 and December 1999 were enrolled in this
study. All patients had thrombocytopenia (platelet count <50 x
109/L) persisting longer than 6 mo, normal or
increased bone marrow megakaryocytes without morphological evidence for
dysplasia, and no secondary immune or nonimmune diseases that could
account for the thrombocytopenic state (1). All patients
received laparoscopic splenectomy because they were refractory to or
could not tolerate corticosteroid therapy. The clinical response to
splenectomy was assessed from the platelet count at 6 mo after
splenectomy. Responders, including patients with a complete or partial
response, were defined as having a platelet count 
50 x
109/L, whereas nonresponders were defined as
having a platelet count <50 x 109/L
(13). Peripheral blood samples were obtained on the day of
splenectomy before the operation, and pieces of the spleen (total
weight >10 g) were processed immediately after resection. In seven
IMTP patients, peripheral blood samples were obtained serially at 3
days and 6 mo after splenectomy. Control samples were obtained from
four patients with cancer (two with gastric cancer and two with
esophageal cancer) who required splenectomy as a part of the dissection
of tumor tissues. These spleens were confirmed to be free of tumor
invasion and metastasis by histopathologic examination. All samples
were obtained after the patients gave their written informed consent,
as approved by the Keio University Institutional Review Board (Tokyo,
Japan).
Preparation of gpIIb-IIIa
Human gpIIb-IIIa was purified from outdated platelet concentrates using affinity chromatography (5). Purified gpIIb-IIIa was dialyzed against PBS with 0.5 mM CaCl2 (PBS-Ca) and stored in aliquots at -80°C until use. gpIIb-IIIa modified by treatment with trypsin was prepared and used for T cell stimulation as described previously (5).
Detection of anti-gpIIb-IIIa Abs
IgG and IgM anti-gpIIb-IIIa Abs in plasma, platelet eluates (from 5 x 107 platelets), and culture supernatants were measured by ELISA using affinity-purified gpIIb-IIIa as an Ag, as described elsewhere (4, 5). Ab units were calculated from the OD450, with the calculation results being based on the standard curve obtained from a serial concentration of mAb to gpIIb-IIIa (clone HPL1; Harlan Laboratories, Leicester, U.K.). All samples were tested in duplicate, and the results were calculated as the duplicate mean. Cutoff values for plasma and platelet-associated IgG anti-gpIIb-IIIa Abs were 5.1 and 3.3, respectively, on the basis of the mean plus 3 x SD of 20 samples from healthy individuals.
Flow cytometric analysis
Cell staining was performed using anti-CD3, anti-CD4,
anti-CD8, anti-CD27, anti-Ig 
, anti-Ig 
(BD
PharMingen, San Diego, CA), anti-CD19, anti-CD38
(Sigma-Aldrich, St. Louis, MO), anti-CD138 (Beckman Coulter,
Fullerton, CA), and anti-CD154 (Ancell, Bayport, MN) mAbs. These
mAbs were conjugated to FITC or PE, or were unconjugated. Cells were
analyzed on a FACSCaliber flow cytometer (BD PharMingen) using the
CellQuest software. Immature/mature B cells were defined as
CD19+/surface Ig+
(+ or +) cells while
plasma cells were defined as
CD19-/CD138+ cells
(14).
Cell preparation
PBMCs were isolated from heparinized venous blood using Lymphoprep (Nycomed, Oslo, Norway) density gradient centrifugation, and resuspended in RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Sterile spleen tissue in complete medium was washed twice to remove peripheral blood and crushed with a syringe plunger. After all of the cells were dissociated, the cell suspension was filtered through a nylon mesh and subjected to Lymphoprep density gradient centrifugation. The recovered cells were suspended in complete medium and used as splenocytes. Freshly isolated PBMCs and splenocytes were used in the following experiments unless indicated otherwise.
In some experiments, T cells were isolated from PBMCs and splenocytes by passing them through a nylon wool column twice as described previously (15) and flow cytometric analysis revealed that the T cell fraction contained >95% CD3+ T cells. PBMCs and splenocytes were depleted of CD4+ cells, CD8+ cells, or CD19+ cells by mixing the cells with magnetic bead-conjugated mAbs (Dynal, Oslo, Norway), followed by magnetic removal of the bead-bound cells. CD27+, CD38+, surface Ig+, CD138+, and CD154+ cells were also removed by incubating the cells with mAbs to these molecules, followed by incubation with goat anti-mouse IgG Ab conjugated to magnetic beads (Dynal). After the depletion treatment, <2% of the cells were positive for these markers, except for CD154+ cells, which made up <0.1% of the population.
Quantification of gpIIb-IIIa-reactive T cells
The frequency of gpIIb-IIIa-reactive T cells was estimated using limiting dilution analysis as described previously (16, 17). Briefly, aliquots containing serial numbers of PBMC- or splenocyte-originated T cells (1, 2.5, 5, 10, 25, and 50 x 103) were cultured with irradiated (40 Gy) autologous splenocytes (104) in the presence of modified gpIIb-IIIa (5 µg/ml) for 5 or 7 days. Each aliquot of T cells was dispensed into 96 wells. Control cultures without Ag were also set up in 12 wells. After a final 16-h incubation with 0.5 µCi/well of [3H]thymidine, the cells were harvested and [3H]thymidine incorporation was determined in a TopCount microplate scintillation counter (Packard Instrument, Meriden, CT). A positive well was defined as having cpm greater than three times the mean counts per minute of the 12 control cultures. Based on the assumption that the responding cells were randomly distributed in the culture wells, the frequency of the responding T cells could be estimated according to a Poisson distribution formula. Only data with statistical significance in a single regression model (p < 0.05) were adopted. Because activated T cells showed accelerated proliferation kinetics upon antigenic stimulation (5), T cells proliferating at day 5 were regarded as activated T cells.
In some experiments, effects of CD4+ cell or CD154+ cell depletion on gpIIb-IIIa-induced T cell proliferation in cultures of PBMCs and splenocytes were examined (5). The results were expressed as the percentage of inhibition, which was calculated as the difference between the cpm incorporated in the cultures with and without treatment divided by the counts per minute incorporated in the culture without treatment.
Detection and quantification of gpIIb-IIIa Ab-producing B cells
B cells producing anti-gpIIb-IIIa Ab were detected and quantified using an ELISPOT assay, which was developed for the detection of autoantibody-producing B cells (18). Briefly, polyvinylidene difluoride-bottomed 96-well multititer plates (Millipore, Bedford, MA) were coated with 30 µg/ml of purified gpIIb-IIIa in PBS-Ca. After incubation at 4°C overnight, the plates were washed three times with PBS-Ca and blocked with 1% BSA in PBS-Ca for 1 h at room temperature. Plates coated with 1% BSA in the absence of gpIIb-IIIa were used as a control. The Ag-coated plates were prepared fresh each time. PBMCs or splenocytes (105 cells/well) in complete medium were incubated in the Ag-coated plates at 37°C in a humidified atmosphere of 5% CO2 for 4 h. The membranes were then washed three times with PBS-Ca containing 0.05% Tween 20, and subsequently incubated with alkaline phosphatase-conjugated goat anti-human IgG or IgM (ICN Pharmaceuticals, Aurora, OH) diluted 1/1000 in PBS-Ca at room temperature for 2 h. After the membranes were washed four times with PBS-Ca containing 0.05% Tween 20 and once with PBS-Ca, Ab bound to the membrane were visualized as spots by incubation with nitro blue tetrazolium/5-bromo-4-chloro-indolyl phosphate. The number of spots was counted under a dissecting microscope. Each experiment was conducted in 10 independent wells, and the results represent the mean of the 10 values. The frequency of anti-gpIIb-IIIa Ab-producing B cells was expressed as the number per 105 mononuclear cells (MNCs), that was calculated by subtracting the number of spots on the control membrane coated with BSA alone from the number of spots on the gpIIb-IIIa-coated membrane. In some instances, the frequencies of anti-gpIIb-IIIa Ab-producing cells were expressed as the number per 105 B cells, based on the proportion of B cells, including immature/mature B cells and plasma cells, in the MNCs. To evaluate B cell subsets producing anti-gpIIb-IIIa Ab, PBMCs and splenocytes that were depleted of CD4+, CD8+, CD19+, CD27+, CD38+, CD138+, and surface Ig+ cells were also used in the ELISPOT assay.
In vitro anti-gpIIb-IIIa Ab production with or without Ag stimulation
In vitro production of anti-gpIIb-IIIa Ab in cultures of PBMCs and splenocytes was evaluated as described (5, 6, 11) with some modifications. Briefly, PBMCs or splenocytes (5 x 105/well) were cultured in 500 µl of complete medium in 48-well culture plates with or without Ag stimulation for 10 days. The Ag-induced anti-gpIIb-IIIa Ab synthesis was examined in cultures with modified gpIIb-IIIa in the presence of PWM (1 µg/ml). PBMCs and splenocytes that were depleted of CD4+, CD8+, CD19+, CD38+, and surface Ig+ cells, were also examined. The levels of IgG and IgM anti-gpIIb-IIIa Abs in undiluted culture supernatants were measured by ELISA. All cultures were prepared in duplicate, and the anti-gpIIb-IIIa Ab results represent the mean of duplicate values. In some experiments, the anti-gpIIb-IIIa Ab produced in culture supernatants was absorbed by preincubation with platelets, erythrocytes, or PBMCs obtained from two healthy individuals (5).
Statistical analysis
All comparisons between two groups were tested for statistical significance using Fisher’s two-tailed exact test or the Mann-Whitney test. The correlation coefficient (r) was determined using a single regression model. Significance of serial changes in T and B cell frequencies and Ab levels was assessed using one-way factorial ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical findings and plasma and platelet-associated IgG
anti-gpIIb-IIIa Ab levels in nine IMTP patients and four controls
are summarized in Table I
. The age at
splenectomy tended to be younger for IMTP patients compared with
control patients. The disease duration between diagnosis and
splenectomy varied and ranged from 7 to 221 mo. All patients were on
low-dose corticosteroids (<10 mg prednisolone/day) at the time of
splenectomy. Intravenous Ig was administered as a pretreatment
for splenectomy in all IMTP patients except patient P1, but none of the
patients received a platelet transfusion at the splenectomy. The
platelet count at 1 wk before splenectomy ranged from 7 to 42 x
109/L in the IMTP patients, whereas all the
control patients had normal platelet counts. Based on the platelet
count at 6 mo after splenectomy, seven patients were responders while
two were nonresponders. Plasma and platelet-associated IgG
anti-gpIIb-IIIa Abs were detected in two and eight IMTP patients,
respectively, but in none of the controls. IgM anti-gpIIb-IIIa Ab
was not detected in any of the subjects.
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Limiting dilution analysis was performed to quantify T cells
responsive to gpIIb-IIIa in PBMCs and splenocytes. As shown in Fig. 1A, the frequency of
gpIIb-IIIa-reactive T cells that proliferated at day 7 was
significantly greater in IMTP patients compared with controls in the
peripheral blood (6.4 ± 2.6 vs 2.2 ± 0.8 per
105 T cells; p = 0.02) and in the
spleen (5.2 ± 2.4 vs 1.5 ± 0.4 per
105 T cells; p = 0.007). Contrary
to our initial expectation, there was no difference in the frequency of
gpIIb-IIIa-reactive T cells between PBMCs and splenocytes in IMTP
patients. However, when limiting dilution analysis was conducted in
5-day cultures, the frequency of gpIIb-IIIa-reactive activated T cells
was significantly higher in splenocytes than in PBMCs (3.4 ± 2.0
vs 1.2 ± 0.4 per 105 T cells;
p = 0.004; Fig. 1B).
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A). CD154, a ligand for CD40,
is expressed transiently on T cells upon activation by Ag stimulation
(19). CD4+ cell-depleted or
mock-treated fractions were also analyzed as controls.
gpIIb-IIIa-induced T cell proliferation was completely inhibited by
depletion of CD4+ cells in both PBMC and
splenocyte cultures. In contrast, suppression of the T cell
proliferation by CD154+ cell depletion was
minimal in PBMCs, but prominent in splenocytes. As shown in Fig. 2B, the mean percentage of inhibition of the T cell
proliferation by CD154+ cell depletion in
splenocyte cultures was significantly greater than that in PBMC
cultures (49.1 ± 12.0 vs 13.7 ± 7.1; p =
0.004). The inhibitory effect by CD154+ cell
depletion in splenocyte cultures tended to be smaller in the two
nonresponders than in the seven responders.
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B cells producing anti-gpIIb-IIIa Ab were detected and
quantified using an ELISPOT assay. Fig. 3A illustrates a
representative result. When PBMCs and splenocytes from IMTP patients
were applied to the assay, clear spots were detected on the
gpIIb-IIIa-coated membranes, but were rarely found on the control
membranes coated with BSA alone. The sizes of individual spots were
highly variable, indicating a difference in capacity of individual
cells to secrete anti-gpIIb-IIIa Ab. Large spots were predominately
found in IMTP splenocytes. Using this technique, the frequencies of
anti-gpIIb-IIIa Ab-producing cells were quantified in PBMCs and
splenocytes from nine IMTP patients and four controls (Fig. 3B). The number of IgG anti-gpIIb-IIIa Ab-producing
cells per 105 B cells was greater in IMTP
patients than in controls in the peripheral blood (61.2 ± 24.0 vs
1.3 ± 0.9; p = 0.007) and in the spleen
(77.7 ± 45.3 vs 4.2 ± 2.5; p = 0.007),
although there was no difference between PBMCs and splenocytes in IMTP
patients. The frequencies of IgG anti-gpIIb-IIIa Ab-producing B
cells in PBMCs and splenocytes were significantly correlated with the
platelet-associated IgG anti-gpIIb-IIIa Ab levels in IMTP
responders (r = 0.86, p = 0.002 and
r = 0.83, p = 0.02, respectively). The
frequency of IgM anti-gpIIb-IIIa Ab-producing B cells was <0.5 per
105 MNCs in all samples.
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As shown in Fig. 4A, the
frequencies of gpIIb-IIIa-reactive T and B cells in individual IMTP
patients were significantly correlated with each other within the
peripheral blood and spleen. However, the gpIIb-IIIa-reactive T cell
frequency was not correlated between the peripheral blood and spleen
(p = 0.11), but was significantly correlated
when the two nonresponders were excluded from the analysis
(r = 0.72, p = 0.05; Fig. 4B, left). Similarly, the gpIIb-IIIa-reactive B
cell frequency was not correlated between the peripheral blood and
spleen (p = 0.14), but a significant
correlation was obtained without the two nonresponders
(r = 0.94, p = 0.004; Fig. 4B, right). These findings indicated that the
frequencies of gpIIb-IIIa-reactive T and B cells in the spleen were
relatively lower than in the peripheral blood in the two
nonresponders.
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PBMCs and splenocytes were cultured for 10 days, and the IgG and
IgM anti-gpIIb-IIIa Abs secreted spontaneously into the culture
supernatants were measured. As shown in Fig. 5A, PBMCs from IMTP patients
scarcely produced IgG anti-gpIIb-IIIa Abs, but IMTP splenocytes
spontaneously produced IgG anti-gpIIb-IIIa Abs in vitro. The amount
of IgG anti-gpIIb-IIIa Ab produced in splenocyte cultures was
significantly larger than that in PBMC cultures in IMTP patients
(3.7 ± 2.3 vs 0.3 ± 0.1; p < 0.001). IgM
anti-gpIIb-IIIa Ab was not detected in any of the culture
supernatants. IgG anti-gpIIb-IIIa Ab reactivity could be removed
from the splenocyte culture supernatants by preincubation with normal
platelets, but not by incubation with erythrocytes or PBMCs (data not
shown), indicating that the IgG anti-gpIIb-IIIa Ab produced in
vitro was capable of binding to normal platelets. The amounts of IgG
anti-gpIIb-IIIa Ab produced in vitro in splenocyte cultures were
significantly correlated with the frequency of IgG anti-gpIIb-IIIa
Ab-producing B cells in splenocytes (r = 0.91,
p = 0.0007). IgG anti-gpIIb-IIIa Ab levels produced
in splenocyte cultures in the two nonresponders tended to be lower than
those in the seven responders.
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B). As a result, there was no difference in
the amounts of anti-gpIIb-IIIa Ab produced upon Ag stimulation
between PBMC and splenocyte cultures. The Ag-induced
anti-gpIIb-IIIa Ab production in PBMC cultures was completely
blocked by depletion of CD4+ cells, indicating
that enhancement of the Ab production was CD4+ T
cell-dependent (data not shown). Differentiation stage of anti-gpIIb-IIIa Ab-producing B cells
Because the anti-gpIIb-IIIa Ab produced by the PBMCs and
splenocytes from IMTP patients was exclusively of the IgG isotype, it
is likely that the Ab-producing cells were memory B cells and/or plasma
cells, which had been already activated via a cognate interaction with
activated CD4+ T cells. To determine the
differentiation stage of the anti-gpIIb-IIIa Ab-producing B cells,
ELISPOT and in vitro assays for anti-gpIIb-IIIa Ab production were
conducted using PBMCs and splenocytes that had been depleted of
CD19+ cells (Fig. 6). CD19 is expressed on the majority of
memory B cells but its expression is largely diminished after
differentiation to plasma cells. Depletion of
CD19+ cells from PBMCs resulted in the
complete loss of anti-gpIIb-IIIa Ab-producing cells and in vitro
anti-gpIIb-IIIa Ab production. In contrast, anti-gpIIb-IIIa
Ab-producing B cells were detectable in splenocytes even after the
CD19+ cells were removed. Moreover,
CD19+ cell-depleted splenocytes from six patients
secreted a detectable level of anti-gpIIb-IIIa Ab into the culture
supernatants. The frequency of anti-gpIIb-IIIa Ab-producing cells
and levels of anti-gpIIb-IIIa Ab produced in vitro in
CD19+ cell-depleted splenocytes were
significantly greater than in CD19+ cell-depleted
PBMCs (7.0 ± 4.8 vs 0.3 ± 0.2; p < 0.001
and 1.2 ± 0.9 vs 0.2 ± 0; p = 0.04,
respectively). These findings suggest that the anti-gpIIb-IIIa
Ab-producing cells in the peripheral blood are exclusively
CD19+ memory B cells, whereas those in spleen
include CD19- plasma cells. It was of note that
the depletion of CD19+ cells from splenocytes
resulted in the loss of anti-gpIIb-IIIa Ab-producing cells and in
vitro anti-gpIIb-IIIa Ab production in the two nonresponders,
indicating the absence of anti-gpIIb-IIIa Ab-secreting plasma cells
in the spleen from these patients.
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. In peripheral blood, the
anti-gpIIb-IIIa Ab-producing cells were completely lost in
CD19+ cell-depleted and surface
Ig+ cell-depleted fractions, whereas depletion of
CD38+, CD4+, or
CD8+ cells had no effect. In contrast, the
decrease in the number of anti-gpIIb-IIIa Ab-producing cells after
the depletion of CD19+ cells or surface
Ig+ cells was partial in splenocytes. Depletion
of CD38+ cells from splenocytes resulted in a
slight decrease in number of the Ab-producing cells, and a complete
loss could be achieved by the depletion of both
CD19+ and CD38+ cells.
Results obtained from in vitro splenocyte cultures were principally
concordant with those from the ELISPOT assay, but partial inhibition of
the anti-gpIIb-IIIa Ab production by CD4+
cell depletion was observed exclusively in splenocyte cultures. Because
this phenomenon was not observed in the ELISPOT assay, it is likely
that splenic CD4+ T cells are involved in the
production of anti-gpIIb-IIIa Ab from B cells in in vitro cultures,
even in the absence of exogenous Ag. Similar results were obtained from
two additional IMTP patients, P4 and P8.
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Serial measurement of the frequencies of gpIIb-IIIa-reactive T and B cells
The frequencies of gpIIb-IIIa-reactive T cells and
anti-gpIIb-IIIa Ab-producing B cells in peripheral blood were
serially examined 3 days and 6 mo after splenectomy in seven IMTP
patients, including five responders and two nonresponders (Fig. 8). The frequencies of
gpIIb-IIIa-reactive T and B cells were significantly decreased after
splenectomy in responders (p < 0.001 and
p = 0.004, respectively, by one-way factorial ANOVA),
but unchanged or increased in nonresponders. A decrease in the
frequency of anti-gpIIb-IIIa Ab-producing B cells was detected 3
days after splenectomy, whereas a decrease in gpIIb-IIIa-reactive T
cells was not apparent at this timepoint. The levels of
platelet-associated anti-gpIIb-IIIa Ab were also decreased after
splenectomy in responders (p = 0.03 by one-way
factorial ANOVA), but not in nonresponders.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B), it is possible that
the peripheral blood in IMTP patients serves as a pool of
gpIIb-IIIa-reactive memory T and B cells with the potential for
secreting anti-platelet Ab. Based on the findings in this study, we propose the following process of continual anti-gpIIb-IIIa Ab production in vivo in IMTP patients. gpIIb-IIIa-reactive memory CD4+ T cells in the circulation arrive in the white pulp of the spleen, then migrate to the outer edge of the periarterial lymphatic sheath, where they interact with APCs, including macrophages and dendritic cells, in the adjacent red pulp. gpIIb-IIIa-reactive T cells are then activated upon recognition of Ag peptides, and interact with gpIIb-IIIa-reactive memory B cells that have also arrived from the circulation, resulting in the production of anti-gpIIb-IIIa Ab. T cell-derived IL-6 is expected to play an important role in this process, as shown in our previous study (6). After gpIIb-IIIa-reactive B cells are activated, some differentiate into plasma cells that constitutively secrete a large amount of anti-gpIIb-IIIa Ab, but have a limited lifespan and eventually undergo apoptosis. In contrast, the majority of gpIIb-IIIa-reactive T and B cells are released into the circulation as memory cells. This model is supported by our recent finding that antigenic gpIIb-IIIa fragments recognized by T cells were principally concordant between PBMCs and splenocytes in the majority of IMTP patients (our unpublished observation). The dynamics of the interaction between gpIIb-IIIa-reactive T and B cells controlling the anti-gpIIb-IIIa Ab synthesis account for the primary role of the spleen in the pathogenesis of IMTP.
Because all but one patient received i.v. Ig as a pretreatment for
splenectomy, we have to consider the possible influence of i.v. Ig on
the T and B cell responses to gpIIb-IIIa. The mechanisms for the in
vivo activity of i.v. Ig in IMTP patients remains unclear, although a
variety of explanations have been put forward to account for this
activity, including FcR blockade, attenuation of complement-mediated
platelet destruction, and neutralization of anti-platelet Abs by
anti-idiotype Abs (22, 23). A recent study using a
murine IMTP model has indicated that i.v. Ig mediates its protective
effect by inducing the expression of the inhibitory Fc
RIIB on
macrophages, resulting in suppression of the clearance of the
sensitized platelets (24). In addition, Levy et al.
(25) reported that anti-gpIIb-IIIa Ab levels in IMTP
patients were not decreased, but even increased after i.v. Ig
treatment. These findings imply that i.v. Ig primarily affects platelet
clearance by macrophages, rather than anti-platelet Ab
production.
The two IMTP patients who did not respond clinically to splenectomy had relatively low frequencies of gpIIb-IIIa-reactive T and B cells in the spleen compared with peripheral blood. In addition, gpIIb-IIIa-reactive activated T cells and anti-gpIIb-IIIa Ab-secreting plasma cells were barely detected in the splenocytes from the nonresponders. Although the number of patients examined was small, these findings strongly suggest that the spleen was not the primary site of gpIIb-IIIa-reactive T and B cell activation in these patients and alternate sites for activation are present outside of the spleen. In fact, in nonresponders, the frequencies of circulating gpIIb-IIIa-reactive T and B cells and the levels of platelet-associated anti-gpIIb-IIIa Ab were unchanged or even increased after splenectomy. Bone marrow is the most likely site for anti-gpIIb-IIIa Ab production in these patients, given that the bone marrow cells from IMTP patients have been shown to produce IgG Abs that bind to platelets in vitro (26). Therefore, the clinical response to splenectomy in IMTP patients appears to be dependent on the site of anti-platelet Ab production. In this regard, it has been reported that IMTP patients who have good response to i.v. Ig are likely to similarly respond to splenectomy (27). Because i.v. Ig exerts its therapeutic effect mainly through suppression of macrophage function, this clinical observation strongly suggests that two distinct pathogenic processes, anti-platelet Ab production induced by activated T and B cells and platelet clearance by macrophages, are closely related in IMTP patients.
Use of the ELISPOT assay enabled us to detect and quantify anti-gpIIb-IIIa Ab-producing B cells in PBMCs and splenocytes. The ELISA is a widely used assay for detecting the Ab, but the ELISPOT assay has several unique features. First, the ELISPOT assay is able to detect even a single cell of 105 cells, whose secretion level may be insufficient for detection by ELISA. In fact, in the majority of IMTP patients, anti-gpIIb-IIIa Ab-producing B cells were detected in PBMCs by the ELISPOT assay, but anti-gpIIb-IIIa Ab was not detectable in PBMC culture supernatants by ELISA. Second, de novo Ab production by B cells can be assessed by the ELISPOT assay, and spot formation may reflect ongoing Ab production in vivo. Regarding this point, the frequencies of anti-gpIIb-IIIa Ab-producing B cells in PBMCs and splenocytes detected by the ELISPOT assay were significantly correlated with the platelet-associated anti-gpIIb-IIIa Ab levels. Finally, the ELISPOT assay can detect the Ab produced by B cells without interference by binding to the Ag. In this regard, the majority of pathogenic anti-platelet Abs in IMTP patients are thought to be present as platelet-associated IgG (28), and it is necessary to use platelets as a source of Ab in the ELISA to detect the anti-gpIIb-IIIa Ab with high sensitivity and specificity (29). Therefore, the ELISA is not widely used for the detection of the anti-gpIIb-IIIa Ab in clinical settings. Because the frequency of circulating anti-gpIIb-IIIa Ab-producing cells was shown to be significantly higher in IMTP patients than in control cancer patients, the ELISPOT assay may be useful clinically for detecting platelet-specific Abs.
How are gpIIb-IIIa-reactive T and B cells activated in the spleen in
IMTP patients? Because circulating memory T and B cells readily
produced anti-gpIIb-IIIa Ab when optimal Ag stimulation was given
in vitro, we presume that the antigenic determinants of gpIIb-IIIa are
efficiently presented by APCs in IMTP spleens. Our previous studies
have shown that gpIIb-IIIa-reactive CD4+ T cells
recognize antigenic peptides generated from chemically modified
gpIIb-IIIa and recombinant fragments produced in bacteria, but not from
gpIIb-IIIa in its native form, suggesting that the epitopes recognized
by gpIIb-IIIa-reactive T cells are cryptic (5). We have
recently found that gpIIb-IIIa-reactive T cells recognize
immunodominant epitopes located within the amino-terminal regions of
both gpIIb
and gpIIIa (6), although the factors that
induce the expression of these cryptic determinants in IMTP patients
are unknown. The de novo presentation of a previously cryptic
self-determinant has been proposed to be induced by up-regulated Ag
presentation capacity and shifts in the peptide hierarchy (30, 31). Because of the association between the process of
anti-platelet Ab production induced by activated T and B cells and
the process of platelet destruction by macrophages in IMTP patients as
mentioned above, it is likely that splenic macrophages that capture
many sensitized platelets play a central role in this process in IMTP
patients. Signaling through FcR would activate the Ag-processing
pathway and up-regulate the expression of adhesion and costimulatory
molecules (32). In addition, splenic macrophages would
have the ability to concentrate and present to T cells a small quantity
of cryptic determinants of gpIIb-IIIa, which would not be efficiently
generated under normal circumstances without platelet sensitization.
The efficient presentation of a small quantity of determinants to T
cells was also reported in cross-reactive B cells that selectively
capture the Ag via the B cell receptor (33). Further
studies examining the mechanisms for activation of gpIIb-IIIa-reactive
T and B cells in the spleen will be useful for clarifying the
pathogenic process in patients with chronic IMTP and for developing a
therapeutic approach that blocks pathogenic anti-platelet Ab
production.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to: Dr. Masataka Kuwana, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address: kuwanam{at}sc.itc.keio.ac.jp
3 Abbreviations used in this paper: IMTP, immune thrombocytopenic purpura; MNC, mononuclear cell.
Received for publication November 13, 2001. Accepted for publication February 4, 2002.
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-receptor antibody. N. Engl. J. Med. 314:1236.[Medline]
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