A study of non-specific stimulation-induced response in B-chronich lymphocytic leukaemia cell line - MEC1 cells

Author: salome oqriashvili
Keywords: Chronic lymphocytic leukaemia, Toll like receptors, CD180
Annotation:

Chronic Lymphocytic Leukemia (CLL) presents with clonal expansion and accumulation of CD5+CD19+CD23+ cells in peripheral lymphoid organs and tissues and bone marrow. The disease can be characterized into two major subgroups, immunoglobulin (Ig) heavy (H) chain gene (IGHV) unmutated and mutated, which is prognostic of an aggressive or an indolent disease respectively. There is increasing evidence that CLL cells receive microenvironmental signals which support their growth, survival and expansion in vivo. Since CLL has been suggested to be antigen driven (although currently unknown) elucidation of both – the antigen itself and the pro-survival signaling pathways are pivotal in identifying a cure for the disease. Possible receptor, which interacts with the microenvironment is a member of Toll-like receptors family – CD180. It has been shown, that CD180 is expressed heterogeneously on CLL cells, and mainly on those with mutated IGHV genes. Ligation of CD180 with monoclonal antibodies (mAb) induced activation of approximately 50% of the CD180+ B-CLL clones, subdividing them into Responders (R) and Non-Responders (NR). CLL cell line MEC1 has been established in 1999 from peripheral blood lymphocytes of EBV-seropositive CLL patient. Since this time MEC1 cell line is approved as a model for CLL immunopathology studies. It should be noted, that MEC1 cell line was established from the patient with mutated IGVH genes (mutation level – 94.6%). At the previous stage of the research, performed by our group, it has been demonstrated, that MEC1 cells are anergic towards B cell receptor (BCR) ligation and can be observed as a model of non-responder CLL clone. Our group also has shown, that MEC1 cells express CD180, but the percentage of CD180-expressing cells isn’t stable: an increase in CD180+ cells number can be seen during exponential growth stage, first 72 hours after the cell culture re-seeding. Taking into account, that anergic MEC1 cells proliferate in response to non-specific stimulation – culture re-seeding and also based on data published by others, that it was possible to induce proliferation of non-responder clones from CLL patients by mitogenic stimulation, we suggested, that MEC1 cells stimulation with mitogen, might give us possibility in case of mitogen-derived proliferation further investigate CD180 participation in pro-survival pathways. Therefore the main aim of our research was to investigate an effect of non-specific stimulation in MEC1 cells. For stimulation we have used phorbolmyristate acetate (PMA). The current research suggested to investigate how mitogen-stimulation affects: 1. Phenotypic profile; 2. Pro-survival pathways; 3. Proliferation process; 4. Activation level. Also additionally we have added a task (5th): to investigate time-related changes in phenotype of MEC1 cells in spontaneous culture. At the previous stage of the research, we already have seen, that during the time interval 24 h – 144h after culture re-seeding, CD180 expression on MEC1 is variable at different time-points. As a control unstimulated cultures re-seeded simultaneously with the experiment have been used. At different time-points we measured proliferation, apoptosis and activation levels in stimulated cultures, also checking surface expression of receptors functionally linked to BCR signalling pathway: CD180 and CD32. Surface expression level was detected by using subsequent fluorochrome-conjugated monoclonal antibodies (mAbs), according standard immunophenotyping procedure. Samples were analysed on flow cytometer (FACScan, Becton Dickinson). To evaluate the proliferative state of cells in response to stimulation, we looked at cells distribution between cell cycle stages by staining of double-stranded DNA with ethidium bromide. To evaluate pro-survival processes, we measured apoptosis level, looking at both: percentage of cells on late stages of apoptosis (on EB staining-derived picture – hypoploid section) and percentage of cells on early stages of apoptosis (using detection of phosphatidylserine surface expression by Annexin V, combined with propidium iodide staining). To evaluate cells activation we checked surface expression of the activation marker – CD38. In case of all abovementioned assays, sample analyses has been performed on flow cytometer (FACScan, Becton Dickinson). According the obtained results an increase in CD180 surface expression level has been detected after the stimulation with PMA (CD180+ cells % spont: 10.4±2.3, PMA-stim.: 30.5±4.6, p<0.01). CD32 expression was unchanged compared to control. At the same time in PMA-stimulated cultures an increased percentage of late apoptotic cells have been detected (cells % in subG0: spont. 15.25±3.9; stim.. 30.43±3.9, p<0.05). From the other side, the percentage of cells at the early stage of apoptosis not only wasn’t increased, but also had a tendency to decrease compared to spontaneous cultures (spont. 13.5±4%; stim 3.31±1.4%). The abovementioned discrepancy can be explained by activation-induced cell death (ACID), which seems to be triggered by PMA in a part of MEC1 cells population. Therefore due to the immediate character of ACID process, at 0.5h time-point all cells already exhibit signs of late apoptosis and can’t be detected by Annexin V/ PI double staining. MEC1 cells distribution between cell cycle stages hasn’t revealed proliferation in response to mitogenic stimulation (cells % in S phase: spont.5±0.7; stim. 3.25±0.5; cells % in G2/M: spont. 2±0.6; stim. 1.9±0.5). While normal lymphocytes respond to PMA with an intensive proliferation. Neither an increase of activation level (according CD38 surface expression) has been detected (CD38+ cells % spont. 30.6±8.1; stim 19.7±5.5 ). In spontaneous cultures, CD38 expression was maximal at 0.5h after re-seeding (30.6±8.1%), while at later time-points it decreased (at 96h: 5±0.01). CD180 expression profile also has been studied in 0.5 – 24h interval after re-seeding. It has been shown, that CD180 expression starts to increase at 8h after re-seeding ( CD180+ cells %: 0.5 h - 10.4±2.3; 8h - 24.2±3.5, p<0.05), reaching maximal point at 24h (29.5±5.2, p<0.05). BCR signalling pathway’s negative regulator – CD32 expression in contrast to CD180 is low at 24h (11±3.8%), while increasing at 96h (36±1.3% ). Abovementioned results reveal that MEC1 cells appeared to be anergic not only towards BCR stimulation, but also towards strong mitogenic stimulation. From the other side with a certain delay a change in phenotyping profile in response to PMA stimulation has been detected: CD180 expression increased on 24h after stimulation. This fact suggests, that beside acting through the same signalling pathway, BCR and CD180 are partially independently regulated. As it has been already mentioned, MEC1 cells present a model for non-responder CLL clone, therefore in case of this anergic clone we have shown, that by an external stimulus can induce modulation of CD180 surface expression. The obtained results will help to investigate the anergy phenomenon of non-responder clones and also the role of CD180 in CLL pathogenesis.

Lecture files:

B ქრონიკული ლიმფოციტური ლეიკემიის უჯრედული ხაზის MEC1 უჯრედებში არასპეციფიური სტიმულაციით გამოწვეული საპასუხო რეაქციის შესწავლა [ka]