Hydroxychloroquine inhibits calcium signals in T cells: a new mechanism to explain its immunomodulatory properties

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3460-3466 ◽  
Author(s):  
Frederick D. Goldman ◽  
Andrew L. Gilman ◽  
Clay Hollenback ◽  
Roberta M. Kato ◽  
Brett A. Premack ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Antigen Receptor ◽  
Calcium Signal ◽  
Dependent Manner ◽  
Tcr Signaling ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Immunomodulatory Properties

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 γ1, 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.

Hydroxychloroquine inhibits calcium signals in T cells: a new mechanism to explain its immunomodulatory properties

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3460-3466 ◽  
Author(s):  
Frederick D. Goldman ◽  
Andrew L. Gilman ◽  
Clay Hollenback ◽  
Roberta M. Kato ◽  
Brett A. Premack ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Antigen Receptor ◽  
Calcium Signal ◽  
Dependent Manner ◽  
Tcr Signaling ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Immunomodulatory Properties

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 γ1, 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.

Rosmarinic acid inhibits Ca2+-dependent pathways of T-cell antigen receptor-mediated signaling by inhibiting the PLC-γ1 and Itk activity

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3534-3542 ◽  
Author(s):  
Mi-Ae Kang ◽  
Su-Young Yun ◽  
Jonghwa Won
Keyword(s):  
T Cells ◽  
T Cell ◽  
Signaling Pathways ◽  
Tyrosine Phosphorylation ◽  
Rosmarinic Acid ◽  
Antigen Receptor ◽  
Tcr Signaling ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen

Rosmarinic acid (RosA) is a hydroxylated compound frequently found in herbal plants and is mostly responsible for anti-inflammatory and antioxidative activity. Previously, we observed that RosA inhibited T-cell antigen receptor (TCR)– induced interleukin 2 (IL-2) expression and subsequent T-cell proliferation in vitro. In this study, we investigated in detail inhibitory mechanism of RosA on TCR signaling, which ultimately activates IL-2 promoter by activating transcription factors, such as nuclear factor of activated T cells (NF-AT) and activating protein-1 (AP-1). Interestingly, RosA inhibited NF-AT activation but not AP-1, suggesting that RosA inhibits Ca2+- dependent signaling pathways only. Signaling events upstream of NF-AT activation, such as the generation of inositol 1,4,5-triphosphate and Ca2+ mobilization, and tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) were strongly inhibited by RosA. Tyrosine phosphorylation of PLC-γ1 is largely dependent on 3 kinds of protein tyrosine kinases (PTKs), ie, Lck, ZAP-70, and Itk. We found that RosA efficiently inhibited TCR-induced tyrosine phosphorylation and subsequent activation of Itk but did not inhibit Lck or ZAP-70. ZAP-70–dependent signaling pathways such as the tyrosine phosphorylation of LAT and SLP-76 and serine/threonine phosphorylation of mitogen-activated protein kinases (MAPKs) were intact in the presence of RosA, confirming that RosA suppresses TCR signaling in a ZAP-70–independent manner. Therefore, we conclude that RosA inhibits TCR signaling leading to Ca2+ mobilization and NF-AT activation by blocking membrane-proximal events, specifically, the tyrosine phosphorylation of inducible T cells kinase (Itk) and PLC-γ1.

scholarly journals ZAP-70-Independent Ca2+ Mobilization and Erk Activation in Jurkat T Cells in Response to T-Cell Antigen Receptor Ligation

2001 ◽  
Vol 21 (21) ◽  
pp. 7137-7149 ◽  
Author(s):  
Xiaochuan Shan ◽  
Richard Balakir ◽  
Gabriel Criado ◽  
Jason S. Wood ◽  
Maria-Cristina Seminario ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Antigen Receptor ◽  
Jurkat Cells ◽  
Jurkat T Cells ◽  
T Cell Antigen Receptor ◽  
Erk Activation ◽  
Cell Antigen Receptor ◽  
Cell Antigen

ABSTRACT The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-γ1 (PLCγ1) and increased the intracellular Ca2+ concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCγ1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.

scholarly journals Differential T-Cell Antigen Receptor Signaling Mediated by the Src Family Kinases Lck and Fyn

2000 ◽  
Vol 20 (4) ◽  
pp. 1426-1435 ◽  
Author(s):  
Michael F. Denny ◽  
Barbara Patai ◽  
David B. Straus
Keyword(s):  
Signal Transduction ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Antigen Receptor ◽  
Alternative Form ◽  
Tcr Signaling ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Tcr Signal Transduction

ABSTRACT Src family tyrosine kinases play a key role in T-cell antigen receptor (TCR) signaling. They are responsible for the initial tyrosine phosphorylation of the receptor, leading to the recruitment of the ZAP-70 tyrosine kinase, as well as the subsequent phosphorylation and activation of ZAP-70. Molecular and genetic evidence indicates that both the Fyn and Lck members of the Src family can participate in TCR signal transduction; however, it is unclear to what extent they utilize the same signal transduction pathways and activate the same downstream events. We have addressed this issue by examining the ability of Fyn to mediate TCR signal transduction in an Lck-deficient T-cell line (JCaM1). Fyn was able to induce tyrosine phosphorylation of the TCR and recruitment of the ZAP-70 kinase, but the pattern of TCR phosphorylation was altered and activation of ZAP-70 was defective. Despite this, the SLP-76 adapter protein was inducibly tyrosine phosphorylated, and both the Ras–mitogen-activated protein kinase and the phosphatidylinositol 4,5-biphosphate signaling pathways were activated. TCR stimulation of JCaM1/Fyn cells induced the expression of the CD69 activation marker and inhibited cell growth, but NFAT activation and the production of interleukin-2 were markedly reduced. These results indicate that Fyn mediates an alternative form of TCR signaling which is independent of ZAP-70 activation and generates a distinct cellular phenotype. Furthermore, these findings imply that the outcome of TCR signal transduction may be determined by which Src family kinase is used to initiate signaling.

scholarly journals Aggregation of Lipid Rafts Accompanies Signaling via the T Cell Antigen Receptor

1999 ◽  
Vol 147 (2) ◽  
pp. 447-461 ◽  
Author(s):  
Peter W. Janes ◽  
Steven C. Ley ◽  
Anthony I. Magee
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Lipid Rafts ◽  
Antigen Receptor ◽  
Cross Linking ◽  
Tcr Signaling ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Associated Proteins

The role of lipid rafts in T cell antigen receptor (TCR) signaling was investigated using fluorescence microscopy. Lipid rafts labeled with cholera toxin B subunit (CT-B) and cross-linked into patches displayed characteristics of rafts isolated biochemically, including detergent resistance and colocalization with raft-associated proteins. LCK, LAT, and the TCR all colocalized with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine phosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-linking of either CD3 or CT-B strongly induced tyrosine phosphorylation and recruitment of a ZAP-70(SH2)2–green fluorescent protein (GFP) fusion protein to the lipid patches. Also, CT-B patching induced signaling events analagous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules. Targeting of LCK to rafts was necessary for these events, as a nonraft- associated transmembrane LCK chimera, which did not colocalize with TCR patches, could not reconstitute CT-B–induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation.

Antigen activation of murine T cells induces tyrosine phosphorylation of a polypeptide associated with the T cell antigen receptor

Cell ◽  
1986 ◽  
Vol 46 (7) ◽  
pp. 1083-1090 ◽  
Author(s):  
Lawrence E. Samelson ◽  
Maitray D. Patel ◽  
Allan M. Weissman ◽  
Joe B. Harford ◽  
Richard D. Klausner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Antigen Receptor ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen

scholarly journals Signaling capacity of the T cell antigen receptor is negatively regulated by the PTP1C tyrosine phosphatase.

1996 ◽  
Vol 184 (3) ◽  
pp. 839-852 ◽  
Author(s):  
G Pani ◽  
K D Fischer ◽  
I Mlinaric-Rascan ◽  
K A Siminovitch
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Phosphatase ◽  
Antigen Receptor ◽  
Sh2 Domain ◽  
Cell Physiology ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen

The association of PTP1C deficiency with the multiplicity of lymphoid cell abnormalities manifested by motheaten (me) and viable motheaten (me(v)) mice suggests a pivotal role for this tyrosine phosphatase in the regulation of lymphocyte differentiation and function. To delineate the relevance of PTP1C to T cell physiology, we have examined me and me(v) T cells with regards to their capacity to transduce activating signals through the T cell antigen receptor (TCR). Although thymocyte maturation appeared normal in the mutant mice, both thymocytes and peripheral T cells from these animals exhibited proliferative response to TCR stimulation that were markedly increased relative to those elicited in normal cells. Compared to normal thymocytes, PTP1C-deficient thymocytes also showed increased constitutive tyrosine phosphorylation of the TCR complex and enhanced and prolonged TCR-induced tyrosine phosphorylation of the TCR-zeta and CD3-epsilon, as well as a number of cytosolic proteins, most notably a 38-kD phosphoprotein found to associate with the Grb2 adaptor SH2 domain in activated thymocytes. These latter phosphoproteins also associated with the Vav guanine nucleotide exchange factor upon TCR ligation, and were dephosphorylated by recombinant PTP1C in vitro. In conjunction with the finding of PTP1C-TCR association in unstimulated normal thymocytes, these results reveal the capacity of PTP1C to interact with and likely dephosphorylate resting and activated TCR complex components, as well as more distal signaling effectors that are normally recruited to the Vav and Grb2 SH2 domains after TCR stimulation. These data therefore strongly implicate PTP1C in the downregulation of TCR signaling capacity and, taken together with the aberrant prolongation of TCR-induced, mitogen-associated kinase (MAPK) activation observed in PTP1C-deficient thymocytes, these findings suggest that the inhibitory influence of PTP1C on TCR signal relay is realized through its effects on both the TCR complex and downstream signaling elements that couple the activated antigen receptor to the Ras/MAPK response pathway.

scholarly journals Regulation of T-cell antigen receptor signalling by Syk tyrosine protein kinase.

1997 ◽  
Vol 17 (8) ◽  
pp. 4434-4441 ◽  
Author(s):  
S Latour ◽  
M Fournel ◽  
A Veillette
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Antigen Receptor ◽  
Gammadelta T Cells ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Tcr Signalling

T-cell antigen receptor (TCR) signalling has been shown to involve two classes of tyrosine protein kinases: the Src-related kinases p56(lck) and p59(fyr), and the Zap-70/Syk family kinases. Lck and FynT are postulated to initiate TCR-triggered signal transduction by phosphorylating the CD3 and zeta subunits of the TCR complex. This modification permits the recruitment of Zap-70 and Syk, which are presumed to amplify the TCR-triggered signal, by phosphorylating additional intracellular proteins. While Zap-70 is expressed in all T cells, Syk is present in thymocytes and mature T-cell populations such as intraepithelial gammadelta T cells and naive alphabeta T cells. To better understand the role of Syk in these cells, its impact on the physiology of an antigen-specific T-cell line was tested. Our results showed that compared to Zap-70 alone, Syk was a strong positive regulator of antigen receptor-induced signals in BI-141 cells. Surprisingly, they indicated that, like Src family kinases, Syk augmented TCR-triggered tyrosine phosphorylation of CD3/zeta. Syk, but not Zap-70 alone, could also stimulate tyrosine phosphorylation of a zeta-bearing chimera in transiently transfected Cos-1 cells. Finally, evidence was provided that Syk has the capacity to directly phosphorylate a zeta-derived peptide in vitro. These findings suggested that Syk may have a unique role in T cells, as a consequence of its ability to efficiently phosphorylate multiple components of the TCR signalling cascade. Furthermore, they raised the possibility that Syk can regulate the initiation of TCR signalling, by promoting phosphorylation of the immunoreceptor tyrosine-based activation motifs of the TCR complex.

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