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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3460-3466
IMMUNOBIOLOGY
Hydroxychloroquine inhibits calcium signals in T cells: a new
mechanism to explain its immunomodulatory properties
Frederick D. Goldman,
Andrew L. Gilman,
Clay Hollenback,
Roberta M. Kato,
Brett A. Premack, and
David J. Rawlings
Department of Pediatrics, University of Iowa, Iowa City; Department
of Pediatrics, Children's Mercy Hospital, University of Missouri-KC,
School of Medicine, Kansas City; Department of Pediatrics, the Jonsson
Comprehensive Cancer Center, and the Molecular Biology Institute and
Department of Physiology, University of California, Los Angeles.
 |
Abstract |
Hydroxychloroquine (HCQ), a lysosomotropic amine, is an
immunosuppressive agent presently being evaluated in bone marrow
transplant patients to treat graft-versus-host disease. While its
immunosuppressive properties have been attributed primarily to its
ability to interfere with antigen processing, recent reports
demonstrate HCQ also blocks T-cell activation in vitro. To more
precisely define the T-cell inhibitory effects of HCQ, the authors
evaluated T-cell antigen receptor (TCR) signaling events in a T-cell
line pretreated with HCQ. In a concentration-dependent manner, HCQ
inhibited anti-TCR-induced up-regulation of CD69 expression, a distal
TCR signaling event. Proximal TCR signals, including inductive protein
tyrosine phosphorylation, tyrosine phosphorylation of phospholipase C
 1, and total inositol phosphate production, were unaffected by HCQ.
Strikingly, anti-TCR-crosslinking-induced calcium mobilization was
significantly inhibited by HCQ, particularly at the highest
concentrations tested (100 µmol/L) in both T-cell lines and primary T cells. HCQ, in a dose-dependent fashion, also reduced a B-cell antigen receptor calcium signal, indicating this effect may be a general property of HCQ. Inhibition of the calcium signal correlated directly with a reduction in the size of
thapsigargin-sensitive intracellular calcium stores in HCQ-treated
cells. Together, these findings suggest that disruption of
TCR-crosslinking-dependent calcium signaling provides an additional
mechanism to explain the immunomodulatory properties of HCQ.
(Blood. 2000;95:3460-3466)
© 2000 by The American Society of Hematology.
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Introduction |
Graft-versus-host disease (GvHD) is a significant cause
of morbidity and mortality in allogeneic bone marrow transplantation. While the pathogenesis of GvHD is not fully understood, activated T
cells are likely to mediate this process. In GvHD, T-cell activation occurs following recognition of host antigens through the T-cell antigen receptor (TCR) on donor T cells. Many pharmacologic agents used
to prevent and treat GvHD inhibit T-cell activation and/or remove T
cells from the circulation. These agents include glucocorticoids, cyclosporin A (CsA), anti-thymocyte globulin, and methotrexate. These
drugs have been used alone or in combination with varying degrees of
success, and each is associated with potentially significant side
effects owing to systemic toxicity.
Hydroxychloroquine (HCQ) is a 4-aminoquinoline antimalarial drug that
has been used for many years to treat autoimmune
disorders.1 HCQ exhibits relatively limited systemic
toxicity with extended use at doses at or below 6.5 mg/kg per day, or
up to 12 mg/kg per day for 2 years. A recent clinical trial suggests
this agent may be useful for the treatment of chronic
GvHD.2 The immunosuppressive properties of HCQ relate, in
part, to its interference with normal antigen processing and
presentation. In vitro, HCQ appears to alter antigen processing by
increasing the pH of intracellular vacuoles, resulting in dissociation
of the invariant chain from the major histocompatibility complex class
II molecule and inhibition of antigen binding.3 More
recently, HCQ has also been shown to block proliferative responses to
T-cell mitogens and alloantigens and to reduce the release of various
cytokines, including interleukin (IL)-6 and tumor necrosis
factor.4,5 These recent findings suggest
that HCQ may function as an immunosuppressant by blocking one or more
steps in the T-cell-activation pathway.
TCR engagement with antigen, or experimentally with anti-TCR
antibodies, results in the initiation of a signal transduction cascade
(or cascades) leading to T-cell activation. The earliest measurable
biochemical events in this cascade are activation of nonreceptor
protein tyrosine kinases (PTKs), including p56lck, p59fyn, and ZAP-70.
This is followed by tyrosine phosphorylation of multiple intracellular
substrates, including SLP-76, LAT, VAV, SHC, and phospholipase C (PLC)
1.6,7 Phosphorylation of PLC1 enhances its catalytic
activity, leading to hydrolysis of phosphatidylinositol
4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and
diacylglycerol.8 These "second messengers" lead to an
increase in intracellular calcium and activation of protein kinase C
(PKC), respectively. Elevated intracellular calcium levels are crucial
for the activation of calmodulin, which binds to and activates the
serine phosphatase calcineurin. Calcineurin, a primary target of CsA,
controls the nuclear translocation of transcription factors of the NFAT
(nuclear factor of activated T cells) family.9 Downstream
events following NFAT activation include up-regulation of CD69 (an
early activation marker) and production and secretion of IL-2.
The present study was undertaken to determine if the immunomodulatory
properties of HCQ are mediated through inhibition of specific
TCR-crosslinking-dependent signaling events. We demonstrate that HCQ,
in a dose-dependent manner, inhibits TCR-mediated calcium mobilization
and other downstream events, including CD69 expression. In contrast,
calcium-independent proximal TCR signaling events, including inductive
protein tyrosine phosphorylation, mitogen-associated protein (MAP)
kinase phosphorylation, and production of inositol phosphates, appear
to be unaffected by HCQ. Because the site of action of HCQ is distinct
from that of other immunosuppressive agents, HCQ may have synergistic
properties when used in combination with other immunosuppressants,
leading to an enhanced anti-GvHD effect.
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Materials and methods |
Reagents
Hydroxychloroquine was kindly provided by Sanofi Pharmaceuticals
(New York, NY) and solubilized in sterile water. Phorbol 12-myrisate
13-acetate (PMA) (Sigma, St Louis, MO) was used at 50 ng/mL, while thapsigargin (Gibco/BRL, Coon Rapids, MN)
was used at 1 µmol/L. Anti-TCR monoclonal antibody
(mAb) C305 ascites, clonotypic for the TCR expressed on Jurkat cells,
was generously provided by A. Weiss (University of California, San
Francisco). Commercial antibodies included anti-CD69 (Leu23a) (Becton
Dickinson, Mountain View, CA), biotinylated anti-CD3 (OKT3)
(PharMingen, San Diego, CA), fluorescein isothiocyanate
(FITC)-conjugated goat antimouse secondary antibody (Cappel, West
Chester, PA), polyclonal antiphosphotyrosine antibody (4G10) (Upstate
Biotechnology, Lake Placid, NY), polyclonal rabbit antirat ERK-2
(Upstate Biotechnology), mixed mouse monoclonal antibovine PLC1
(Upstate Biotechnology), rabbit and goat antimouse immunoglobulin
(Jackson Immunoresearch Laboratories, West Grove, PA), and a
horseradish peroxidase (HRPO)-conjugated rabbit antimouse antibody
(Bio-Rad, Chicago, IL).
Cells
Jurkat T cells (clone E6-1) and Ramos B cells (American Type Culture
Collection, Rockville, MD) were maintained in complete RPMI
medium (10% fetal calf serum, penicillin 1000 U/mL, streptomycin 1000 U/mL, and
glutamine 20 mmol/L). Human peripheral blood
mononuclear cells (PBMCs) from healthy volunteers were isolated by
Ficoll-Hypaque gradient centrifugation. Following isolation, cells were
plated at a density of 1 to
2 × 106/mL and left in tissue
culture flasks at 37°C in 5% CO2 for 2 hours to remove
adherent cells.
Flow cytometry and CD69 expression
Jurkat cells were pretreated with HCQ for 24 hours and then
stimulated with RPMI alone (control), PMA (50 ng/mL), or C305 (1:2000) for an additional 24 hours.
Cells were then washed and stained with FITC-conjugated anti-CD69, and
fluorescence intensity was measured on a FACScan (Becton Dickinson).
Analysis of intracellular free calcium concentration
Jurkat T cells or Ramos B cells were loaded with the
calcium-sensitive indicator dye calcium green-1-AM
(C-3012) (Molecular Probes, Eugene, OR) 5 µg/mL, in RPMI (10 mmol/L
HEPES, 2% fetal calf serum) for 30 minutes at
37°C. Excess dye was washed away with Hanks' Balanced Salt
Solution (HBSS) supplemented with 1.3 mmol/L CaCl2 supplemented with 10 mmol/L
HEPES). Loaded cells were resuspended in HBSS and kept at 4°C and
protected from light prior to analysis. Intracellular calcium
measurements were monitored by the use of 530 nm
fluorescence emission following 506 nm excitation by means of a bulk
spectrofluorimeter (Photon Technology International, Monmouth Junction,
NJ). Following 20 seconds of data acquisition, 5 × 105 Jurkat/mL
was activated by the addition of anti-TCR mAb C305 (1:2000 dilution of ascites fluid), and Ca++ mobilization
was monitored. Ramos B cells were activated by addition of 10 µg/mL of goat anti-immunoglobulin (Ig)M antibody
(2020-01) (Southern Biotech, Birmingham, AL).
Residual peak calcium response was evaluated by addition of 1 µmol/L ionomycin. Thapsigargin-sensitive calcium
stores were evaluated in the presence of 4 mmol/L
egtazic acid (EGTA) by addition of 1 µmol/L
thapsigargin (586005) (Calbiochem, San Diego, CA). For
calcium studies in primary human T cells, HCQ-treated or untreated
PBMCs (5 × 107/mL) were loaded with
calcium green-1-AM and fura-red-AM (F3021) (Molecular
Probes) at a final dye concentration of 10 µmol/L for 30 minutes at 37°C. Loaded cells were washed once in calcium buffer (25 mmol/L HEPES, 125 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L
Na2HPO4, 0.1% glucose, 500 µmol/L MgCl2, 10 mmol/L
CaCl2, 0.1% bovine serum albumin, pH 7.4), resuspended at
1.5 × 106/mL in calcium buffer, and
immediately placed on ice. Prior to activation, cells were warmed to
37°C for 15 minutes. Following 30 seconds of data acquisition,
cells were activated by addition of biotinylated anti-CD3 (40 µg/mL), followed after 10 seconds by avidin (10 µg/mL). The calcium green versus fura-red
fluorescence ratio of individual cells was measured by means of a
FACScan flow cytometer (Becton Dickinson) before and after the addition
of activators and analyzed by means of the FlowJo FACS software. Data
are presented in arbitrary units as a function of fluorescence (relative intracellular calcium) versus time.
Notably, initial measurements of intracellular calcium made with the
UV-light-excitable calcium indicator dyes fura-2 or indo-1 were found
to be unreliable owing to interference of HCQ with either the
excitation or the emission properties of these dyes (data not shown).
Briefly, with the use of fura-2-loaded Jurkat cells, excitation scan
analysis of HCQ-treated cells indicated a significantly reduced
absorbance, between approximately 300 nm and 350 nm. Because the
calcium-bound excitation occurs at approximately 340 nm, this artifact
led to an underestimate of intracellular free Ca++
concentrations. With the use of indo-1-loaded cells, emission scan
analysis of HCQ-treated cells (or of free HCQ in HBSS) indicated that
HCQ emits intensely from approximately 375 nm to 425 nm. Because the
calcium-bound emission of this indicator occurs at approximately 400 nm, this artifact would lead to an overestimate of intracellular free
Ca++. In contrast to these findings, HCQ did not
interfere with either calcium-bound or unbound excitation or emission
spectra of either calcium green-1 or fura-red (data not
shown). For this reason, calcium green-1, or calcium green-1 and
fura-red, were used as the indicator dyes for all the experiments described.
Measurement of soluble inositol phosphate generation
Jurkat cells were incubated with various concentrations of HCQ for
24 hours, then loaded with [3H] myoinositol (40 µCi/mL) (Amersham, Arlington Heights, IL) for 3 hours
in phosphate-buffered saline. Duplicate samples were stimulated with
media alone (control) or C305 (1:2000) for 10 minutes, then lysed in a
1:1 mixture of chloroform/methanol. Soluble inositol phosphates were
extracted and separated by anion exchange as previously described and
were quantified by liquid scintillation counting.10
Immunoprecipitation and Western blotting
Following stimulations, Jurkat cells were pelleted, supernatant
aspirated, and 1× lysis buffer (1% NP-40, 150 mmol/L NaCl, 20 mmol/L Tris base, pH
7.4) containing protease (50 mg/mL aprotinin, 10 mg/mL leupeptin, 50 mg/mL pepstatin A,
1 mmol/L PMSF) and phosphatase (400 mmol/L sodium orthovanadate, 10 mmol/L sodium fluoride, 10 mmol/L
sodium pyrophosphate) inhibitors was added to a final concentration of
5 × 107 cells/mL. Samples were left
on ice for 30 minutes and were centrifuged at 12 000g for 15 minutes, and supernatant was saved. For immunoprecipitation of PLC1,
40 µL of protein A (Sigma) was prearmed with
5 µL rabbit antimouse immunoglobulin overnight at
4°C. The prearmed protein A and 1 µL of
anti-PLC1 were added simultaneously to the clarified lysate and
rotated for 2 hours at 4°C. Immunoprecipitated complexes were
washed 5 times in ice-cold lysis buffer. For PLC1
immunoprecipitates, a fraction was immunoblotted with either
anti-PLC1 (5 × 106 cell equivalents) or
antiphosphotyrosine (2 × 107 cell equivalents). In
experiments examining inductive protein tyrosine phosphorylation or
phosphorylation of MAP kinase, whole-cell lysates were prepared by
pelleting rapidly 106 cells and lysing in 1× sodium
dodecyl sulfate sample (SDS) buffer. Immunoprecipitates or proteins
from whole-cell lysates were subjected to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred electrophoretically to
nitrocellulose paper. Nitrocellulose membranes were blocked 2 hours in
a Tris-buffered saline solution containing 3% bovine serum albumin,
and primary antibodies were added for 4 hours at 4°C.
Immunoreactive bands were visualized by means of enhanced
chemiluminescence (Amersham) or BCIP/NBT (Promega, Madison, WI)
according to the manufacturer's instructions.
| |
Results |
HCQ inhibits TCR-activation-induced CD69 expression
Initial experiments were undertaken in Jurkat cells, a human
leukemic T-cell line, to determine whether HCQ alters downstream TCR-signal-transduction-dependent events. Cells were preincubated for
24 hours in the presence of various concentrations of HCQ, stimulated
an additional 24 hours with clonotypic anti-TCR mAb (C305) or phorbol
esters (PMA), then assayed for up-regulation of CD69 by flow cytometry.
Up-regulation of CD69 provides a quantitative measure of T-cell
activation and occurs 12 to 24 hours following TCR ligation or PKC
activation with PMA.11,12 In non-HCQ-treated control
cells, C305 up-regulated CD69 receptor expression, as indicated by an
approximately 0.5 log shift in mean fluorescence intensity compared
with untreated controls (Figure 1).
Treating cells with increasing concentrations of HCQ resulted in a
60%-80% reduction in the expression of CD69 following anti-TCR
stimulation. In contrast, PMA markedly up-regulated CD69 receptor
expression (~1.5 log shift; Figure 1, lower panel), although HCQ
treatment had minimal effect on this TCR-independent event. Notably,
HCQ also exerted no appreciable effect on cell viability during the course of these experiments, even at high concentrations (100 µmol/L
HCQ), and no alterations were noted in the level of
cell surface expression of TCR/CD3, CD4, and CD45 (data not shown).
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| Fig 1.
HCQ down-regulates TCR-induced CD69 expression.
Jurkat T cells were cultured for 24 hours with varying concentrations
of HCQ, then incubated an additional 12 hours in 24-well plates
(1 × 106/mL) in medium alone
(dotted lines), PMA (50 ng/mL), or anti-TCR mAb C305
(ascites, 1:2000). Cells were then harvested and stained with
FITC-conjugated anti-CD69, and fluorescence intensity was measured on a
FACScan. Cell counts and viability were indistinguishable in untreated
and HCQ-treated cell populations. Representative data from more than 5 similar experiments are shown.
|
|
HCQ does not inhibit anti-TCR-induced protein tyrosine kinase
activation, tyrosine phosphorylation of PLC1, or inositol phosphate
production
The observation that HCQ significantly inhibited TCR-dependent CD69
up-regulation but did not interfere with PMA-induced CD69 expression
suggested a block in the TCR signal transduction cascade. We initially
determined if HCQ-mediated inhibition of TCR signals was a consequence
of limited PTK activation or of a reduced capacity to phosphorylate
critical protein substrates. To assay PTK activation, proteins from
detergent lysates of resting or anti-TCR-stimulated Jurkat cells were
separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted
with antiphosphotyrosine antibodies. Pretreating cells with HCQ for 24 hours did not appreciably alter the magnitude, global pattern, or
kinetics of anti-TCR-induced protein tyrosine phosphorylation (Figure
2 and data not shown). These results
suggested that the PTKs operative in proximal TCR signaling were
largely intact in HCQ-treated cells.
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| Fig 2.
HCQ does not inhibit TCR-mediated inductive protein
tyrosine phosphorylation.
Jurkat T cells (1 × 106) were treated with varying
concentrations of HCQ for 24 hours, stimulated with C305 for 5 minutes,
and then lysed in NP-40 detergent. Proteins from whole-cell lysates
were separated by 10% SDS-PAGE, transferred to nitrocellulose, and
immunoblotted with antiphosphotyrosine antibody (4G10). Immunoreactive
bands were visualized by enhanced chemiluminescence. Representative
data from more than 3 similar experiments are shown. A more extended
kinetic analysis of protein tyrosine phosphorylation in untreated and
HCQ-treated cell populations gave similar results (data not shown).
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Tyrosine phosphorylation of PLC1 following TCR engagement enhances
its intrinsic enzymatic activity, leading to increased production of
IP3 and release of Ca++ from IP3-receptor-gated calcium
stores.13,14 To determine whether HCQ altered PLC1
activation, Jurkat cells were pretreated with varying concentrations of
HCQ for 24 hours, stimulated with C305 for 15 minutes, and then lysed.
PLC1 immunoprecipitates from detergent lysates were subjected to
Western blotting with either anti-PLC1 or antiphosphotyrosine
antibodies. Equivalent amounts of PLC1 were immunoprecipitated from
both untreated and HCQ-treated cells (Figure
3, lower panel). Inductive tyrosine phosphorylation of PLC1 was also unaltered following pretreatment with HCQ (Figure 3, upper panel). To indirectly investigate PLC1 enzymatic activity, HCQ-treated Jurkat cells were stimulated with C305,
and total inositol phosphate production was quantified. As shown in
Figure 4, C305 stimulation resulted in an
equivalent increase in total inositol phosphates in both untreated
Jurkat cells and Jurkat cells pretreated with varying concentrations of
HCQ (stimulation index = ~4-5). In addition, HCQ did not alter C305-induced specific IP3 production as measured by radioreceptor assay
(data not shown). Together, these findings suggest that proximal TCR
signaling events, including PLC1 activation and inositol phosphate
production, are intact in T cells treated with HCQ.
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| Fig 3.
HCQ does not alter TCR-induced PLC1 tyrosine
phosphorylation.
Jurkat T cells were treated with varying concentrations of HCQ for 24 hours, stimulated with C305 for 15 minutes, and then lysed in NP-40
detergent. PLC1 was immunoprecipitated from
5 × 107 cell equivalents, and immunoprecipitates
were blotted with either antiphosphotyrosine (upper panel) or
anti-PLC1 (lower panel), and immunoreactive bands were visualized by
enhanced chemiluminescence. Representative data from more than 3 similar experiments are shown.
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| Fig 4.
HCQ does not inhibit TCR-induced inositol phosphate
production.
Jurkat T cells were treated with varying concentrations of HCQ for 24 hours and then loaded with [3H] myoinositol for 3 hours.
Cells were left untreated (none) or were stimulated with C305 for 15 minutes, and were then lysed in chloroform. Soluble inositol phosphates
were extracted by anion exchange and quantitated by liquid
scintillation counting. Mean data from 3 separate experiments are
expressed as stimulation indices: (cpm stimulated/cpm
unstimulated) ± SD.
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HCQ does not inhibit anti-TCR-induced ras signaling
The ras-dependent signaling pathway in T cells leads to a
calcium-independent activation of the MAP kinase
cascade.15-17 The integrity of this pathway can be
monitored experimentally by the appearance of p42 and p44
phosphorylated MAP kinase species (ERK1 and ERK2) with the use of
anti-ERK Western blotting of TCR-stimulated cell
lysates.18,19 To assess the effect of HCQ on this signaling pathway, Jurkat cells were pretreated with HCQ, stimulated with C305
for 0 to 5 minutes, and then assayed for the appearance of phosphorylated forms of ERK2. In both control cells and cells treated
with varying concentrations of HCQ, TCR stimulation consistently resulted in the appearance of equivalent amounts of p44 ERK2 (Figure 5). These results indicated that HCQ does
not significantly limit the signals leading from TCR to MAP kinase
activation.
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| Fig 5.
HCQ does not alter MAP kinase activation.
Jurkat T cells were treated with varying concentrations of HCQ for 24 hours, stimulated with C305 for 0 to 5 minutes, and then lysed in NP-40
detergent. Proteins from whole-cell lysates were subjected to 12%
SDS-PAGE and were transferred to nitrocellulose, and membranes were
immunoblotted with anti-ERK-2. Immunoreactive bands were visualized by
enhanced chemiluminescence. Representative data from more than 3 similar experiments are shown.
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HCQ inhibits TCR-mediated intracellular calcium flux
TCR crosslinking results in a biphasic calcium flux. This consists
of an initial phase increase in intracellular calcium released from
IP3-receptor-gated stores followed by a secondary, sustained phase of
extracellular Ca++ influx. This latter phase is controlled
primarily through depletion of intracellular calcium
stores.13,14,20-22 To determine if HCQ affects
TCR-dependent calcium signaling, Jurkat cells were treated with varying
concentrations of HCQ for 24 hours and assayed for their ability to
flux calcium. Strikingly, as shown in Figure 6A, total TCR-mediated intracellular
calcium mobilization was inhibited by HCQ in a dose-dependent fashion.
Blunted calcium signaling was consistently observed at concentrations
of 25 µmol/L HCQ and higher. To determine if
HCQ-dependent alterations in calcium responses also occurred in
nontransformed T-cell populations, we evaluated the effect of HCQ
treatment on freshly isolated human T cells from healthy donors. PBMCs
were left untreated or were treated for 18 hours with varying
concentrations of HCQ, loaded with the calcium-sensitive dyes calcium
green-1 and fura-red, and monitored for intracellular Ca++
flux by means of flow cytometry (Figure
7A). Consistent with the effect in Jurkat T
cells, HCQ treatment resulted in a dose-dependent reduction in the
calcium response following CD3 crosslinking with nearly complete loss
of the response to receptor engagement at doses of 100 µmol/L HCQ. Finally, to determine if inhibition of receptor-mediated calcium mobilization is a general property of HCQ, we
also evaluated the effect of HCQ on a B-cell line (Ramos). HCQ
treatment resulted in a significant reduction in the calcium signal
initiated by engagement of the B-cell antigen receptor (BCR). At doses
of 100 µmol/L HCQ, complete inhibition of the intracellular response was observed (Figure 6B).
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| Fig 6.
HCQ inhibits antigen-receptor-dependent intracellular
calcium mobilization and alters the size of intracellular calcium
stores.
Jurkat T cells or Ramos B cells were pretreated with varying
concentrations of HCQ for 24 hours and then loaded with the
calcium-sensitive dye calcium green-1. Changes in intracellular
Ca++ concentration (fluorescence) versus time (seconds)
were monitored by bulk spectrofluorometry. (A) Jurkat T cells were
activated with the clonotypic anti-TCR mAb C305 (as indicated by the
arrow) followed by addition of 1 µmol/L ionomycin
(arrowhead). (B) Ramos B cells were activated by IgM crosslinking
(arrow) followed by the addition of 1 µmol/L
ionomycin (arrowhead). (C) Jurkat cells were stimulated with 1 µmol/L thapsigargin, as indicated by arrow, in the
presence of 1.8 mmol/L EGTA. Residual calcium response
was evaluated by addition of 1 µmol/L ionomycin
(arrowhead). Results are representative of more than 5 separate
experiments.
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| Fig 7.
HCQ inhibits intracellular calcium mobilization in
primary T cells.
Peripheral blood T cells from healthy volunteers were pretreated with
varying concentrations of HCQ for 18 hours and were then loaded with
calcium-sensitive dyes calcium green-1 and fura-red. Cells were
stimulated, as indicated by arrow, with (A) biotinylated OKT3 (40 µg/mL) plus avidin (10 µg/mL) or
(B) 1 µmol/L ionomycin. Changes in intracellular
Ca++ concentration (fluorescence) versus time (seconds)
were monitored by fluorescence-activated cell sorter, and results are
representative of 3 separate experiments.
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The inhibition of intracellular calcium mobilization in HCQ-treated
cells correlated with a reduced response following addition of the
calcium ionophore, ionomycin (arrowhead in Figure 6A-C, arrow in Figure
7B). Ionomycin-dependent calcium release was reduced after both TCR and
BCR activation, and the reduction of this response correlated with the
HCQ dose. These findings suggested that the intracellular calcium store
size was altered in HCQ-treated cells. Eukaryotic cells sequester
Ca++ within intracellular calcium stores, including dynamic
Ca++ stores capable of both rapid Ca++ release
in response to IP3-receptor engagement and rapid Ca++
re-uptake via sarcoplasmic or endoplasmic reticulum
Ca++-ATPases (SERCA) pumps.14,23,24 The
sesquiterpene lactone, thapsigargin, selectively inhibits
Ca++ re-uptake via SERCAs.25,26 In the absence
of extracellular calcium, thapsigargin can be used to block the
refilling of these intracellular calcium stores and measure their
relative size without initiation of store-operated calcium
influx.27,28 We therefore evaluated the potential effects
of HCQ on the relative size of this dynamic calcium store in Jurkat T
cells. Strikingly, thapsigargin-sensitive intracellular calcium stores
were significantly reduced in cells pretreated with HCQ. This effect
was HCQ-dose-dependent and was observed both before and after TCR
crosslinking (Figure 6C, and data not shown). Together, these data
demonstrate that HCQ treatment significantly reduces TCR-dependent
calcium signaling and that this effect is controlled, at least in part,
by alteration in the size of the IP3-gated intracellular calcium store.
| |
Discussion |
Quinolines are a group of drugs with newly recognized
immunosuppressive properties. Recent studies have suggested that HCQ may be useful in the treatment of chronic GvHD in humans.2 In addition, the closely related drug chloroquine can prevent acute
GvHD in murine models.29 As diprotic bases, these agents pass through the lipid cell membrane and preferentially concentrate in
acidic cytoplasmic vesicles. The resulting pH change within these
vesicles in macrophages disrupts antigen processing, a mechanism of
action previously proposed to explain the immunomodulatory properties
of these drugs.30 More recently, both HCQ4,31 and chloroquine32 have been shown to have T-cell-specific
effects. These agents inhibit tetanus-toxoid- and mitogen-induced
proliferative responses in vitro and have been used in combination with
CsA to synergistically inhibit T-cell responses in
vitro.31,32
To better understand how HCQ inhibits T-cell activation, we evaluated
the effects of this agent at various points in the TCR signal
transduction cascade. Consistent with previous studies suggestive of
T-cell-specific inhibitory effects,4,31 HCQ inhibited
TCR-induced up-regulation of CD69. Assessment of multiple proximal
TCR-mediated signaling events, including TCR-induced PTK activation,
inositol phosphate production, and MAP kinase activation, failed to
demonstrate an inhibitory effect of HCQ. In contrast, TCR-induced
intracellular calcium mobilization was significantly inhibited by HCQ
in a dose-dependent fashion. Both TCR-crosslinking-dependent initial
calcium release (peak response) and extracellular calcium influx
(sustained response) were inhibited by HCQ. Most notably, HCQ
pretreatment was associated with a dose-dependent reduction in
thapsigargin-sensitive intracellular calcium stores. TCR-crosslinking-dependent depletion of these stores activates the
calcium-release-activated calcium (ICRAC) channel in T
cells, leading to a sustained influx of extracellular
calcium.21 Together, our data suggest that HCQ limits both
the initial peak calcium response and store-operated calcium influx via
ICRAC channels by altering thapsigargin-sensitive,
endoplasmic reticulum calcium store size.
The reduction in the TCR-dependent CD69 up-regulation in HCQ-treated
cells is consistent with previous studies suggesting a role for a
sustained calcium signal (in addition to ras/MAP kinase activation) in
this transcriptional response.12 Consistent with this
model, activation of Jurkat T cells with both PMA and ionomycin leads
to a significant increase in CD69 induction relative to PMA alone (data
not shown). The consequences of altered calcium signaling in B and T
lymphoid cells have been extensively characterized. Both peak threshold
and sustained increases in [Ca++]i are
critical for the induction of the transcription factors NF-AT and
NF- B and for T-cell proliferation and differentiation in response to
TCR crosslinking.33-36 In B cells, subthreshold oscillations in [Ca++]i result in activation
of only a subset of downstream transcription signals and can lead to
anergy and/or cell death via apoptosis.37,38 Because
calcium-dependent signaling events in T and B cells are highly
conserved, subthreshold calcium signals are likely to similarly down-regulate T-cell transcriptional events. Interestingly, BCR-induced intracellular calcium mobilization is also inhibited by HCQ in a
dose-dependent fashion. This inhibition also correlates with a
reduction in the size of thapsigargin-sensitive calcium stores (R.M.K.
and D.J.R., unpublished observations). While the consequences of these
changes on B-cell activation and differentiation are currently being
evaluated, these findings suggest HCQ treatment may alter TCR-, BCR-,
and T- and B-dependent functional immune responses at several levels.
The mechanism by which HCQ exerts its effects on calcium store size
remains unclear. Our data are consistent with either a reduced store
capacity, reduced store refilling, or a combination of both effects.
Previous studies of the effect of chloroquine on G-protein-coupled
receptor-dependent calcium signaling suggested reduced size of
thapsigargin-sensitive calcium stores in fibroblast lines and
macrophages.39 Furthermore, chloroquine was also shown to
alter the capacity of IP3-gated receptors to bind IP3 in these cells.
These data are difficult to interpret, however, because they used the
calcium indicator dye Fura-2. This indicator leads to an underestimate
of intracellular calcium concentration because of interference with the
calcium-bound excitation spectra (see "Material and methods"). If
we assume that similar results would be obtained with an alternative
(eg, calcium green-1, or calcium green-1 and fura-red) measurement,
these earlier studies are consistent with our results demonstrating
reduced intracellular calcium mobilization in HCQ-treated cells. In
both situations, this alteration in calcium signaling occurred despite
equivalent levels of PLC1 activation and IP3 production in
drug-treated and untreated cells. Finally, it has also been reported
that chloroquine can reduce calcium currents in other cell types,
including Paramecium40 and frog atrial
trabeculae.41 Given the similar structures of these
pharmacologic agents, it is not surprising that HCQ could exert similar
effects on calcium currents in human T cells.
Patients receiving HCQ in a trial for treatment of chronic GvHD have
been targeted to have a whole-blood level of 5 to 15 µmol/L. While this "therapeutic" concentration
of HCQ did not consistently inhibit TCR-induced calcium responses in
vitro, we were able to observe a modest but consistent decrease in
TCR-induced CD69 up-regulation at these concentrations. Our
observations that HCQ inhibits TCR-mediated signals in vitro may
partially explain its ability to control chronic GvHD. Because the
mechanism of action of HCQ differs from that of other
immunosuppressants, including CsA and glucocorticoids, it may be well
suited to complement these agents in combination drug therapy for
chronic GvHD. Additional studies are required to fully characterize the
effect of HCQ on calcium signaling in T cells and to assess the
TCR-signaling pathway in T cells from patients with chronic GvHD
receiving HCQ.
| |
Acknowledgments |
We wish to thank Drs Gary Koretzky and Gail Bishop for their critical
review and useful suggestions, Justin Fishbaugh for flow cytometry
assistance, and Marsha Jensen for preparation of this article.
| |
Footnotes |
Submitted July 22, 1999; accepted January 21, 2000.
Supported by National Institutes of Health (NIH) grant
5P30HD27748; by the Carver Medical Trust Funds and grant IN-122Q
from the American Cancer Society, administered through the University of Iowa Cancer Center (F.D.G.); and in part by NIH grants HD37091 and
AR01912, the American Cancer Society, and the facilities of the UCLA
Jonsson Comprehensive Cancer Center (D.J.R.). D.J.R is recipient of
a McDonnell Scholar Award; a Leukemia Society of America Scholar Award;
UCLA Child Health, HHMI, and Pennington Research Awards; and the Joan
J. Drake Grant for Excellence in Cancer Research.
Reprints: Frederick D. Goldman, Department of
Pediatrics, Division of Hematology/Oncology, University of Iowa
Hospitals and Clinics, Iowa City, IA 52242; e-mail:
frederick-goldman{at}uiowa.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Zvaifler NJ.
Update in rheumatology: focus on hydroxychloroquine.
Am J Med.
1988;75:68.
2. Gilman A, Morris C, Mogul M, et al. Hydroxychloroquine for the
treatment of chronic graft-versus-host disease [abstract]. Biol Blood
Bone Marrow Transplant. 2000. In press.
3.
Lombard-Platlet S, Bertolino P, Deng H, Gerlier D, Rabourdine-Combe C.
Inhibition by chloroquine of the class II histocompatibility complex-restricted presentation of endogenous antigens varies according to the cellular origin of the antigen-presenting cells, the nature of the T-cell epitope, and the responding cell.
Immunology.
1993;80:566[Medline]
[Order article via Infotrieve].
4.
Gilman AL, Beams F, Tefft M, Mazumder A.
The effect of hydroxychloroquine on alloreactivity and its potential use for graft-versus-host disease.
Bone Marrow Transplant.
1996;17:1069[Medline]
[Order article via Infotrieve].
5.
Sperber K, Quraishi H, Kalb TH.
Selective regulation of cytokine secretion by hydroxychloroquine: inhibition of interleukin-1-alpha (IL-1- ) and IL-6 in human monocytes and T cells.
J Rheumatol.
1993;20:803[Medline]
[Order article via Infotrieve].
6.
Chan AC, Desai DM, Weiss A.
The role of protein tyrosine kinases and protein tyrosine phosphatases in T cell antigen receptor signal transduction.
Annu Rev Immunol.
1994;12:555[Medline]
[Order article via Infotrieve].
7.
Wardenburg JB, Fu C, Jackman JK, et al.
Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.
J Biol Chem.
1996;271:19,641[Abstract/Free Full Text].
8.
Nishibe S, Wahl MI, Hernandez-Sotomayor SMT, Tonks NK, Rhee SG, Carpenter G.
Increase of the catalytic activity of phospholipase C gamma 1 by tyrosine phosphorylation.
Science.
1990;250:1253[Abstract/Free Full Text].
9.
Rao A, Luo C, Hogan PG.
Transcription factors of the NFAT family: regulation and function.
Annu Rev Immunol.
1997;15:707[Medline]
[Order article via Infotrieve].
10.
Koretzky GA, Picus J, Schultz T, Weiss A.
The tyrosine phosphatase CD45 is required for T cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production.
Proc Natl Acad Sci U S A.
1991;88:2037[Abstract/Free Full Text].
11.
Hara T, Jung L, Bjorndahl J, Fu SM.
Human T cell activation, III: rapid induction of a phosphorylated 28 kD/32 kD disulfide-linked early activation antigen (EA-1) by TPA, mitogens, and antigens.
J Exp Med.
1986;164:1988[Abstract/Free Full Text].
12.
Testi R, Phillips JH, Lanier LL.
T cell activation via Leu-23 (CD69).
J Immunol.
1989;142:1123.
13.
Gardner P.
Calcium and T lymphocyte activation.
Cell.
1989;59:15[Medline]
[Order article via Infotrieve].
14.
Berridge MJ.
Inositol trisphosphate and calcium signalling.
Nature.
1993;361:315[Medline]
[Order article via Infotrieve].
15.
Williams NG, Paradis H, Agarwal S, Charest DL, Pelech SL, Roberts TM.
Raf-1 and p21v-ras cooperate in the activation of mitogen-activated protein kinase.
Proc Natl Acad Sci U S A.
1993;90:5772[Abstract/Free Full Text].
16.
Franklin RA, Tordai A, Patel H, Gardner AM, Johnson GL, Gelfand EW.
Ligation of the T cell receptor complex results in activation of the ras/raf-1/MEK/MAPK cascade in human T lymphocytes.
J Clin Invest.
1994;93:2134.
17.
Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ, Johnson GL.
A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf.
Science.
1993;260:315[Abstract/Free Full Text].
18.
DeVries-Smits AMM, Burgering BMT, Leevers SJ, Marshall CJ, Bos JL.
Involvement of p21ras in activation of extracellular signal-regulated kinase 2.
Nature.
1992;357:602[Medline]
[Order article via Infotrieve].
19.
Leevers SJ, Marshall CJ.
Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein.
EMBO J.
1992;11:569[Medline]
[Order article via Infotrieve].
20.
Hoth M, Penner R.
Depletion of intracellular calcium stores activates a calcium current in mast cells.
Nature.
1992;355:353[Medline]
[Order article via Infotrieve].
21.
Premack BA, McDonald TV, Gardner P.
Activation of Ca2+ current in Jurkat T cells following the depletion of Ca2+ stores by microsomal Ca(2+)-ATPase inhibitors.
J Immunol.
1994;152:5226-5240[Abstract].
22.
Misra UK, Chu CT, Rubenstein DS, Gawdi G, Pizzo SV.
2-Macrogloboin receptor-recognized forms elevate intracellular calcium and cyclic AMP in murine peritoneal macrophages.
Biochem J.
1993;291:885.
23.
Bootman MD, Berridge MJ.
The elemental principles of calcium signaling.
Cell.
1995;83:675[Medline]
[Order article via Infotrieve].
24.
Clapham DE.
Calcium signaling.
Cell.
1995;80:259[Medline]
[Order article via Infotrieve].
25.
Thastrup O, Dawson AP, Scharff O, et al.
Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage.
Agents Actions.
1989;27:17[Medline]
[Order article via Infotrieve].
26.
Lytton J, Westlin M, Hanley MR.
Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.
J Biol Chem.
1991;266:17,067[Abstract/Free Full Text].
27.
Gouy H, Cefai D, Christensen SB, Debre P, Bismuth G.
Ca2+ influx in human T lymphocytes is induced independently of inositol phosphate production by mobilization of intracellular Ca2+ stores: a study with the Ca2+ endoplasmic reticulum-ATPase inhibitor thagsigargin.
Eur J Immunol.
1990;20:2269[Medline]
[Order article via Infotrieve].
28.
Takemura H, Hughes AR, Thastrup O, Putney JW Jr.
Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells: evidence that an intracellular calcium pool and not an inositol phosphate regulates calcium fluxes at the plasma membrane.
J Biol Chem.
1989;264:12,266[Abstract/Free Full Text].
29.
Schultz KR, Bader S, Paquet J, Wei L.
Chloroquine treatment affects T-cell priming to minor histocompatibility antigens and graft-versus-host disease.
Blood.
1995;86:1[Free Full Text].
30.
Fox R.
Anti-malarial drugs: possible mechanisms of action in autoimmune disease and prospects for drug development.
Lupus.
1996;1(suppl):S4.
31.
Schultz KR, Nelson D, Bader S.
Synergy between lysosomotropic amines and cyclosporin A on human T cell responses to an exogenous protein antigen, tetanus toxoid.
Bone Marrow Transplant.
1996;18:625[Medline]
[Order article via Infotrieve].
32.
Landewe RBM, Miltenburg AMM, Breedveld FC, Daha MR, Dijkmans BAC.
Cyclosporine and chloroquine synergistically inhibit the interferon-gamma production by CD4 positive and CD8 positive synovial T cell clones derived from a patient with rheumatoid arthritis.
J Rheumatol.
1992;19:1353[Medline]
[Order article via Infotrieve].
33.
Ho SN, Thomas DJ, Timmerman LA, Li X, Francke U, Crabtree GR.
NFATc3, a lymphoid-specific NFATc family member that is calcium-regulated and exhibits distinct DNA binding specificity.
J Biol Chem.
1995;270:19,898[Abstract/Free Full Text].
34.
Hivroz-Burgaud C, Clipstone NA, Cantrell DA.
Signaling requirements for the expression of the transactivating factor NF-AT in human T lymphocytes.
Eur J Immunol.
1991;21:2811[Medline]
[Order article via Infotrieve].
35.
Kerschbaum HH, Negulescu PA, Cahalan MD.
Ion channels, Ca2+ signaling, and reporter gene expression in antigen-specific mouse T cells.
J Immunol.
1997;159:1628[Abstract].
36.
Kanno T, Siebenlist U.
Activation of nuclear factor-kappaB via T cell receptor requires a Raf kinase and Ca2+ influx: functional synergy between Raf and calcineurin.
J Immunol.
1996;157:5277[Abstract].
37.
Healy JI, Dolmetsch RE, Timmerman LA, et al.
Different nuclear signals are activated by the B cell receptor during positive versus negative signaling.
Immunity.
1997;6:419[Medline]
[Order article via Infotrieve].
38.
Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI.
Differential activation of transcription factors induced by Ca2+ response amplitude and duration.
Nature.
1997;386:855[Medline]
[Order article via Infotrieve].
39.
Misra UK, Gawdi G, Pizzo SV.
Chloroquine, quinine and quinidine inhibit calcium release from macrophage intracellular stores by blocking inositol 1,4,5-trisphosphate binding to its receptor.
J Cell Biochem.
1997;64:225[Medline]
[Order article via Infotrieve].
40.
Barry SR, Bernal J.
Antimalarial drugs inhibit calcium-dependent backward swimming and calcium currents in Paramecium calkinsi.
J Comp Physiol [A].
1993;172:457[Medline]
[Order article via Infotrieve].
41.
Filippov A, Skatova G, Porotikov V, Kobrinsky E, Saxon M.
Ca2+-antagonistic properties of phospholipase A2 inhibitors, mepacrine and chloroquine.
Gen Physiol Biophys.
1989;8:113[Medline]
[Order article via Infotrieve].
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Abstract
Introduction