Effects Of Maltose-binding Protein On Nonspecific Activation Of T Cells And Hematologic Cancer Cells

Maltose-binding protein (MBP) is a high affinity protein responsible for the capture andtransportation of maltose/maltodextrin through the periplasmic space in Gram-negativebacteria. In molecular biology research, MBP is common fused with interest proteins toimprove their yield and facilitate purification. It endowed target proteins with additionalproperties of enhanced stability and solubility. Given the inert or minimum bioactivity,MBP is usually utilized to fuse with interest proteins in various experimental subunitvaccines against pathogenic bacteria and viruses. However, recent study showed that MBPinduced human dendritic cell (DC) maturation and its proinflammatory cytokines productionvia TLR4activation. We found that fusion MUC1with MBP could enhance itsimmunogenicity and induced the cell immune response. MBP enhanced the immuno-stimulatory activity of vaccines in coordination with BCG in a tumor-bearing mouse model.We also found that MBP triggered activation of macrophage, induced the differentiation ofM1macrophage, and induced the activation of NK cells. These studies provide evidence thatMBP has immunopotentiating properties in enhancement of immune responses. It hopes thatMBP will be one of adjuvant family members. However, the mechanism of MBP regulatedimmune response is still unclear.The aim of present study is to investigate whether immunocytes are activated and whichsort of immunocyte is activated in murine spleen when stimulated with MBP, and to furtherexplore the biological effects of MBP on tumor cells in vitro and its underlying mechanism.一.The mechanism of MBP nonspecific activation of T lymphocytes1. MBP promotes the proliferation of the mouse spleen lymphocytesWe utilized MTT to detect the effect of MBP on the mouse splenocytes, and the studyshowed that MBP promoted the proliferation of the mouse spleen lymphocytes in dose andtime dependent manner, anti-MBP antibody could block the effect of MBP on cells. Theresults indicated that MBP promoted the proliferation of the mouse spleen lymphocytes.2. MBP promotes the proliferation of T lymphocytesWe utilized FACS to detect the ratio of CD3+and CD19+cells in mouse spleenlymphocytes after stimulated with MBP, finding that the proportion of CD3+cells were relatively elevated whereas the CD19+cells reduced. The results indicated that MBPpromoted the proliferation of T lymphocytes.3. MBP induces the activation of Th1cellsThe levels of IL-2, IFN-γ and IL-4in the cell supernatants were detected by ELISA, theresults showed that MBP significantly enhanced secretion of IL-2and IFN-γ, whereassecretion of IL-4slightly inhibited; We utilized FACS to detect the effect of MBP on theproportion of secretion IFN-γ and IL-4cells in CD3+and CD4+cells, finding that theproportion of secretion IFN-γ cells in CD3+and CD4+cells were enhanced, suggesting thatMBP induced the activation of Th1cells.4. MBP affects the lymphocytes directlyWe utilized immunofluorescence and immunohistochemistry staining to detect theeffect of MBP on the mouse splenocytes. In immunofluorescence staining positive cells withfluorescence were observed under fluorescence microscope. MBP–binding cells as positivecells became brown in immunohistochemistry staining, and almost all positive cells werelymphocytes and its ratio was about37.7±8.9%in all lymphocytes. After the lymphocyteswas neutralized beforehand with anti-MBP antibody at proper dilution, the positivelymphocytes could not be nearly found, suggesting that MBP directly affected thelymphocytes.5. MBP and Con A synergistically promote mouse lymphocyte proliferationAfter treatment with MBP and Con A, cell proliferation was assessed using WST assay.The results suggested that MBP and Con A synergistically promoted mouse lymphocyteproliferation. We utilized FACS to detect the effect of MBP on the proportion of CD3+andCD19+cells and secretion IFN-γ and IL-4cells in CD3+and CD4+cells. The results showedthat the proportion of CD3+cells and secretion IFN-γ cells in CD3+and CD4+cells wereunchanged compared with Con A group(P>0.05), suggesting that MBP and Con Asynergistically promoted mouse lymphocyte proliferation.二. The mechanism of the effects of MBP on U937and Jurkat cells1. MBP promotes the proliferation of U937and Jurkat cellsWe used WST and blocking experiment to observe the role of MBP in cells. The resultsshowed that MBP enhanced the viability of U937and Jurkat cells in dose and timedependent manner. To further verify our results on the MBP-mediated cell viability, wecharacterized cell cycle distribution in response to1μg/ml MBP for0,24,48,72, and96h.DNA profile analysis revealed an accumulation of U937and Jurkat cells in an S-phase concomitant, and a decrease in G0/G1and G2/M events. To determine whether U937andJurkat cell viability was mediated by MBP, we used anti-MBP mAb to block MBP before itspresence, and cell viability was inhibited by the blocker of MBP.2. MBP directly affects U937and Jurkat cellsWe utilized immunofluorescence and immunohistochemistry staining to detect theeffect of MBP on U937and Jurkat cells. In immunohistochemistry staining, MBP–bindingcells as positive cells became brown to be observed. In immunofluorescence stainingpositive cells with fluorescence were observed under fluorescence microscope. MBP wasable to directly bind to U937and Jurkat cells and suggested that MBP directly affected thecells.3. MBP induces U937cell differentiationTo examine the outcome in U937and Jurkat cells, we assessed the MBP-mediatedeffects on cellular morphology. After48h treatment with1μg/ml MBP, U937cells withpseudopodia were examined by phase contrast light microscopy. Morphological changes ofU937cells were detected by Wright-Giemsa staining, and similar results were obtained.Jurkat cells did not vacuolate the cytoplasm and showed very few changes.4. U937and Jurkat cells modulate TLR expression in response to MBP stimulationMBP activated signaling pathway for DC maturation via TLR4. The resultsdemonstrated that MBP could bind to the cell surface, therefore, we investigated whether theeffects of MBP on U937and Jurkat cells were also via TLRs. We first examined thecell-surface TLR expression patterns in U937and Jurkat cells. Next, cells were either leftuntreated or treated with1μg/ml MBP for the indicated time and RT-PCR was performedunder nonsaturating conditions for cell-surface TLRs. RT-PCR analysis of β-actinexpression confirmed the quality of all RNA preparations used for RT-PCR. TLR1,-2,-4,-5,and-6expression was detected in U937cells, however, all except for TLR2expression weremodulated after MBP stimulation. Only TLR1,-2,-5, and-6were present in Jurkat cells,and the TLR1,-5, and-6expressions were regulated after treatment with MBP. TLR4expression was not observed in both untreated and treated Jurkat cells. MBP regulated TLRexpression in U937and Jurkat cells in the mRNA level. We further observed the TLR2andTLR4expression in U937and Jurkat cells in the protein level. We found that TLR2and TLR4expression was up-regulated with MBP stimulation in U937cells, but TLR4expression was down-regulated in Jurkat cells, and TLR2expression was not observed inJurkat cells. We further examined the effects of anti-TLR2and anti-TLR4treatment onMBP-induced viability of cells to characterize the membrane receptor of MBP. TheMBP-mediated viability of U937cells was inhibited with anti-TLR2, but not anti-TLR4. Theblocking of TLR2and TLR4had no effect on Jurkat cell viability in response to MBPtreatment. Suggesting that U937and Jurkat cells modulated TLR expression in response toMBP stimulation, and MBP mediates U937cell viability via TLR2.5. MBP induces NF-κB and MAP kinase activation in U937and Jurkat cellsMyD88is an important adaptor protein required by all TLRs with the exception ofTLR3, and it is recruited to TLRs by a TLR ligand engagement to mediate inflammatoryresponses to microbial components. To examine the role of MyD88in the MBP-mediatedviability responses, we observed the change of MyD88mRNA and protein levels in the cells.MyD88expression was weakly regulated in response to MBP in mRNA level, however,MyD88protein expression was up-regulated in U937cells and down-regulated in Jurkatcells in treatment with MBP. The activation of TLRs results in NF-κB and MAP kinaseactivation. We treated cells with1μg/ml MBP at the indicated time to determine whether theMBP-induced viability responses in the cells were also dependent on NF-κB and MAPkinase activation. The activation of the signaling pathway was measured as phosphorylationof the proteins. MBP was able to induce rapid NF-κB activation as evidence byphosphorylation of NF-κB p65subunit in U937and Jurkat cells. MBP was also able to affectphosphorylation of p38and JNK1/2/3in U937cells, and induce the phosphorylation ofJNK1/2/3in jurkat cells. Constitutive p38MAPK activation was found in Jurkat cells, andgradually decreased with the MBP-stimulated time. Phosphorylation of ERK1/2wasundetected in U937and Jurkat cells.6. MBP improves cell sensitivity to chemotherapeutic drugsMBP stimulated U937and Jurkat cells in24h, then Paclitaxel treated cells in48h. Theresults showed that Paclitaxel significantly inhibited MBP-stimulated cell growth,suggesting that MBP improves cell sensitivity to chemotherapeutic drugs.Collectively, our results demonstrated that MBP could nonspecifically induce the Th1activation to enhance cellular immunity, and MBP could directly promote U937cellproliferation via TLR2, and MBP played a mitogen-like role in Jurkat cells to stimulate the cell proliferation, cells in proliferation status were high sensitive to paclitaxel, suggestingthat MBP may be used as an adjuvant for participating in the immunotherapy of hematologicmalignancies.