Introduction

Trichosanthin (Tk), a plant protein isolated from a Chinese medicinal herb Trichosanthes kirilowii Maximovich, has been used as an abortifacient for several decades in China. Tk is a linear polypeptide of 247 amino acid residues, the encoding gene of which has been cloned and sequenced. As a ribosome inactivating protein (RIP), Tk is believed to exert its biological activity in inducing cytotoxicity1. However, at lower doses, Tk could inhibit HIV replication and suppress in vitro immune responses of both mouse and human lymphocytes2, 3. Several reports have been released dealing with treatment of AIDS with Tk in clinical trails 4. And for some Tk preparations, the HIV inhibition has been reported to be quite free from their toxic property5. The mechanisms of this intriguing phenomenon remains puzzle. In our previous study, we showed that the Tk-induced hyporeaction is irrelevant to direct cytolysis in the dose range used. In the lymphoproliferation to purified protein derivative (PPD), tetanus toxoid (TT) and anti-CD3 McAb, Tk induced suppression with great extent. However, only a little Tk's immunosuppression was observed in PMA-induced proliferation system in presence of Tk. Since the differential effects of PMA and other stimuli in activation of T cells mainly reflect that they use distinguishable signaling pathways, we propose that Tk inhibits T cell activation might be via interference of signal transduction.

Triggering of TCR/CD3 complex activates the complex-coupled intracellular protein tyrosine kinases (PTKs) to phosphorylated phospholipase C, PLCγ1, and the guanine nucleotide binding protein p21ras. The src family PTKs p56lck and p59fyn are thought to be responsible for this early phosphorylation event. The tyrosine phosphorylation of the CD3 andζchain leads to recruitment of the ZAP-70 and Syk tyrosine kinase by binding of their two SH2 domains to the tyrosine-phophorylated immunorecepter tyrosine-activation motif (ITAM) of CD3ζchain. Subsequently, the activated ZAP-70 phosphorylates PLCγ16.

In our previous study, we were able to show that Tk could inhibit soluble or alloantigen-induced lymphoproliferation by a CD8+ T cell-mediated pathway, and this was genetically controlled by HLA-DQ genes3, 7. In this paper, we examined the ZAP-70 and CD3ζchain in human T cells after Tk treatment. Our results indicated that, under the influence of Tk, the intracellular pathway of signal transduction initiated by triggering of TCR/CD3 complex was interrupted in an essential step, the recruitment of ZAP-70 to CD3ζchain.

Materials and Methods

Cell culture

Jurkat leukemia T cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated newborn calf serum, 2 mM L- glutamine, 100U/ml Penicillin and 100 μg/ml Streptomycin. The cells were washed three times with PBS before use. 1 x 107/ml cells were pulsed with 10 μg/ml Trichosanthin (Jingshan Pharmaceutical, Shanghai) for 3 h at 37°C and washed three times with ice-cold PBS. The Tk-pulsed cells were stimulated with anti-CD3 McAb (ATCC, USA) at a dose 10 μg/ml for 5 min and crosslinked with anti-mouse antibody for 5 min. When PTK was assayed for quantitative expression, Tk was subjected to be co -cultured with cells for 24 h.

Immunoblotting

Cells were lysed in lysis buffer (20 mM Tris-HC1 pH 7. 5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 100 mM NaF, 10 mM Na4P2O7, 1 mM Na3VO4) for 30 min at ice-bath. Lysates were precleared by centrifugation at 12,000 × g for 10 min at 4 °C and the postnuclear supernatants were collected. Protein concentration was carefully measured using the Bradford assay8 and the samples were adjusted to be able to load 50 μg on each lane for electrophoresis. Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulus membrane (Schleicher and Schuell, USA) in 20 mM Tris, 150 mM glycine and 20% methanol at 250 mA for 2 h6. The membrane were saturated in blocking buffer (0.1% Tween-20 TBS) with blocking reagent (Amersham, UK) for 2 h at room temperature and then probed with 1:1000 antiphosphotyrosine antibody (6G9, Life Technologies) or 1:1000 anti-ZAP-70 antisera (kindly gifted by Dr. Weiss, A., UCSF), followed by incubation with 1:1000 peroxidase-conjugated rabbit anti-mouse Ig for 1 h, then washed and developed by enhanced chemiluminesence (ECL, Amersham, UK) according to the manufacturer's instructions.

Immunoprecipitation

107cells were stimulated with saturated amounts of anti-CD3 McAb as described above, then pelleted and lysed in lysis buffer. The cells were precleared with protein A sepharose CL-4B 50% w/v slurry. ZAP-70 or CD3ζchain was immunoprecipitated with anti ZAP-70 antibody or anti-CD3ζchain antibody (Santa Cruz, USA). Immunoprecipitaed protein were washed, subjected to 10% SDS-PAGE, and immunoblotted with antiphosphotyrosine antibody or anti-ZAP-70 antibody.

Results and discussion

Inhibition of CD3 McAb-induced T cell proliferation by Tk

We described previously that the soluble antigen-induced lymphoproliferation could be strongly inhibited by Tk in vitro3. When assayed by proliferation of Jurkat leukemia T cells, there was a significant difference observed between the groups with or without stimulation of anti-CD3 antibody. However, if T cells were pulsetreated with Trichosanthin in advance, the difference was disappeared (Fig 1). It suggested that TCR/CD3 complex-initiated signaling could be interrupted by Tk treatment in some way. For a further assessment of the mechanism underlying, some essential links of the pathways in signal transduction have been examined under the influence of Tk. The mobilization of cytosolic calcium driven by combining of TCR/CD3 with anti-CD3 McAb, for example, was firstly observed to be blocked in our experiment (data have been separately submitted for publication). And it has been shown that the translocation of PKCαand the autophosphorylation of PKC was also inhibited by Tk treatment9. These implicates that, in addition to PKC, protein tyrosine phosphorylation may need to be examined.

Fig 1
figure 1

The low response of the Tk-pulsed Jurkat T cell to anti-CD3 McAb stimulation. [T cells were stimulated with (B, D) or without (A, C) anti-CD3 McAb]. no Tk pulsed Tk pulsed

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The profile of antiphosphotyrosine after Tk treatment

Protein tyrosine phosphorylation was critical upon T cell activation. Triggering of TCR/CD3 complex induces the phosphorylation of many PTK-related signal transduction events in T cells10. As shown on the line 3 of Fig 2, the anti-CD3 McAb-activated T cells showed dark bands when their cell lysates were analysed by probing with antiphosphotyrosine antibody. However, if T cells were pretreated with Trichosanthin and then pulsed with anti-CD3 McAb, only some of tyrosine phosphorylation bands could be detected (line 2), which were much weaker than those not only in group treated with CD3 McAb (line 3) but also in the T cells without Tk treatment (line 1). It implicates that the poor tyrosine phosphorylation was resulted from Tk pretreatment.

Fig 2
figure 2

The profile of antiphosphotyrosine after Tk treatment (experiment was repeated three times) lane 1: Jurkat T cell without treatment; lane 2: Tk pulse-treatment following by stimulation with anti-CD3-McAb; lane 3:anti-CD3 McAb stimulation.

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Inhibition of ZAP-70 for tyrosine phosphorylation but not for expression by Tk treatment

To know what kind of phosphArylation-related protein or kinase was effected by Tk, we examined the activation of ZAP-70 kinase by using immunoprecipitation and immunoblotting assay. ZAP-70 was a protein tyrosine kinase which functions at the downstream of CD3ζchain activation12. When TCR/CD3 complex was triggered, the change of structure conformation initiated phosphorylation ofζchain. Phosphorylated CD3ζchain recruited ZAP-70 kinase by interaction between two ITAMs ofζchain and SH2 domain of the kinase12. As first step, the expression intensity of ZAP-70 was determined. Jurkat leukemia T cells were co-cultured with Trichosanthin for 24 h. Cell lysate was dissolved by 10% SDS-PAGE and probed with anti-ZAP-70 antibody. As shown in Fig 3A, the band of 70 kD ZAP in the Tk cultured group shows same darkness as that in control, suggesting that the expression of ZAP-70 were not affected by Trichosanthin. Then, we examined the phosphorylation of ZAP-70. We immunoprecipitated ZAP-70 from cell lysate with anti-ZAP-70 antibody. As detected with antiphosphotyrosine antibody for the phosphorylated tyrosine in ZAP-70 immunoprecipitant, the darkness of phosphotyrosine band in Tk-treated experiment group was weaker than that in normal cells (Fig 3B). The results suggested a decreasing phosphotyrosine after Trichosanthin treatment.

Fig 3
figure 3

The role of Tk on the expression of ZAP-70 (A) and the phosphorylation of ZAP-70 (B). lane 1: T cells; lane 2: Tk-treated T cells (experiment was repeated three times).

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Dissociation between CD3ζchain and ZAP-70 after Tk treatment

Jurkat leukemia T cells were pulsed with Trichosanthin and then stimulated with anti-CD3 McAb for 5 mim. CD3ζchain were immunoprecipitated from cell lysate with rabbit anti-CD3ζchain antibody. This immunoprecipitant was then probed with anti-ZAP-70 antisera. As a results, the ZAP-70 protein band was visualized much less obviously in the CD3ζchain immunoprecipitant of Tk-pulsed cells when compared to that of normal cells (Fig 4). It suggested that the recruitment of ZAP-70 to CD3ζchain was damaged by Trichosanthin pretreatment in TCR/CD3 complex-initiated signal transduction.

Fig 4
figure 4

The role of Tk on the recruitment of ZAP-70 to CD3ζchain. lane 1: T cells; lane 2: Tk-treated T cells (experiment was repeated three times).

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Usually, the studies on RIPs function of Trichosanthin were carried out by cell lysate. Here, we examined its function on intact T cells using the Tk at a dose range much lower than that causing cytotoxicity. According to our results, Tk was capable of blocking the physical link between CD3ζchain and ZAP-70, leading to a dysfunction of T cells. Obviously, the exact mechanism needs to be explored by further experiments. There are some similar studies with regard to the defective TCR-related signaling caused by external agents13, 14. How the extracellular Tk could exert its activity on the intracellular biochemical events of T cells was still not clear although there was evidence presented in our work that Tk was able to enter the antigen-presenting cells by exocytosis15.