Concanavalin A and Phytohaemagglutinin Stimulated Lymphoproliferative Responses in Cord Blood Mononuclear Cells

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1 Concanavalin A and Phytohaemagglutinin Stimulated Lymphoproliferative Responses in Cord Blood Mononuclear Cells FARUK SÜZERGÖZ 1,2, BAYRAM KIRAN 1, GÜNNUR DEN Z 1, M. FAT H EVC M K 1, OSMAN ÇEN 2 1 stanbul University, Institute for Experimental Medical Research. Department of Immunology. Istanbul, Turkey 2 Harran University Art and Science Faculty, Department of Biology, Þanl urfa, Turkey Experimental and clinical evidences have demonstrated that umbilical cord blood is an efficient source of transplantable haematopoietic stem and progenitor cells. After transplantation, initial exposure to alloantigen-presenting cells (allo-apc) induced lymphoproliferative responses in cord blood T cells. However, this response is lower than that by adult T cells. The aim of this study was to investigate and compare lymphoprolifertive responses of cord blood mononuclear cells (CBMC) and adult peripheral blood mononuclear cells (PBMC). CBMC and PBMC immunoproliferative responses were measured by MTT assay after stimulation of respective primary culture cells with phytohaemagglutinin (PHA) or Concanavalin A (Con A) for 76 hours. Lymphoproliferative response of CBMC was significantly lower compared to that of PBMC. Turk J Immunol, 2005; 10: Key Words: Cord blood, Con A, PHA, Lymphoproliferation, GVHD. U INTRODUCTION mbilical cord blood (UCB) as an alternative to bone marrow for haematopoietic stem cell (HSC) transplantation may lower the risk of graftversus-host disease (GVHD) 1,2. UCB from unrelated donor can restore the function of bone marrow and sustain haematopoietic recovery in both related and unrelated recipients 3,4. As compared to bone marrow transplantation (BMT), the major advantages of cord blood transplantation (CBT) is the reduced incidence and severity of GVHD 5. Several immunologic properties and peculiarities of cord blood lymphocytes may be responsible for the reduction of GVHD after CBT 6. In vitro studies of UCB show alterations in immune reactivity, which may explain, although partially the reduced incidence of GVHD 1. Proliferative responses of the UCB T- cells is reduced with low antigen or mitogen stimulation 7,8 The ability to generate alloantigen specific cytotoxic T lymphocytes is also reduced 9,10. UCB serum appears to contain significantly less soluble factors that enhance mitogen and interleukin-2 specific T- cell growth compared to adult serum 11. MATERIALS AND METHODS Patients: Informed consents were taken from all donors according to the institutional regulations (I.U / 053). For PBMCs 10 ml blood was drawn from 14 normal donors. UCB 13

2 TURK J IMMUNOL Vol.10, No.1, 2005 was obtained from 14 mothers at the end of full-term deliveries, in short, 10 ml blood was withdrawn from the placenta just after delivery with sterile syringes and immediately transferred into separate collection tubes containing heparin (Becton Dickinson Cat No: ). Preparation of cells: Mononuclear cells (MNCs) were separated from the blood samples by density gradient centrifugation with Ficoll-hypaque in sterile conditions. In short, the blood samples were diluted at ratio 1:1 in RPMI 1640 (Sigma Cat No: R0883) containing 2 % FCS (Sigma Cat No: F 4135). The diluted samples were layered over Histopaque «(Sigma Cat No: ) at 1:1 ratio and centrifuged at 1800 rpm, 18 ûc for 30 minutes. The midlayer containing MNCs were taken, washed three time with RPMI, and suspended in IMDM (Sigma Cat No: I 2510) in the presence of 10 % FCS. Cell viability and counting: The cell viability was assessed with trypan blue exclusion test. In short, cells were mixed with trypan blue (Sigma Cat No: T 8154) and immediately counted under the microscope (Olympus). The blue cells were considered non-living cells. The percentage of living cells were counted with following formula. % Living cells = No. of living cells Cell No x100 No. of total cells Colorimetric MTT assay: 10 5 cells from each sample were cultured in 100 µl 10 % FCS-IMDM in triplicates in 96 well plates (Nunc ) and incubated at 37ûC, 5 % CO 2 for 72 hrs in the presence (test wells) or absence (control) of Con A (5 µg /ml) (Sigma Cat no: C2010) or PHA (10 µg /ml) (Sigma Cat no: L8902 ). The cells were than added 10 µl of 5 mg / ml MTT (methyl-thiasoletetrazolium, Sigma Cat no: M5655) and continued incubation for 4 more hours. The plates were then centrifugated at 1200 rpm for 10 min and 50 µl of the supernatant was discarded. Then, 50 µl of DMSO (Dimethyl sulfoxide, Sigma Cat No: D2650) was added to each well and incubated at room temperature (at dark) overnight. The spectrophotometnic values of the cultures were obtained in ELISA Plate Reader (Organon Technica Reader 530) at 540 nm using 620 nm as reference filter. % Proliferaion = Mean OD of test wells x100 Mean OD of control wells OD: optic density Statistical analyses: Results are expressed as mean ± SEM. The differences in the proliferative responses of the cell groups were analysed by student-t test. The effects of mitogens on the cells response were correlated to each other by Pearson Correlation. P values less than 0.05 were considered significant. RESULTS Con A and PHA are mitogenic to lymphocytes. In this study, MNCs obtained from either peripheral blood or cord blood of full-term deliveries are stimulated with Con A or PHA and their proliferative responses were assessed with MTT assay. MTT, taken up by cells, is reduced to MTT-formazan by mitochondria of activated cells. MTT formazan is dissolved in DMSO giving a purple colour detectable by spectrophotometer. Therefore, high levels of MTT-formazan is proportionally related to activity level of the cells. The proliferative responses of the cells to the Con A or PHA stimulation is given in Table 1. When stimulated with 5 µg / ml Con A, the proliferative response of PBMCs was increased to % ± 4.80 while that of CBMCs was % ± Similarly, when 14

3 Vol.10, No.1, 2005 TURK J IMMUNOL Table 1: The proliferative responses of PBMC or CBMC in response to mitogens. The lower, upper, and mean values of each cell group to Con A or PHA are given. Cell Con A PHA source Lower Upper Mean ± SEM Lower Upper Mean ± SEM PBMC 80 % 137 % % ± % 143 % % ± 3.93 CBMC 56 % 113 % % ± % 115 % % ± 3.13 stimulated with 10 µg / ml PHA, proliferative response of cells were defined as % ± 3.93 and % ± 3.13, respectively (Table 1 and Figure 1). The changes in the lymphocyte proliferative responses to Con A (p<0.001) were detected to be higher than that of PHA (p<0.034) (Figures 1 and 2). The cellular proliferative responses to both mitogens, Con A and PHA, were found to demonstrate a positive correlation (r:510, <0.008) with Pearson Correlation (Figure 3). Although, statistically not different, PBMCs show higher proliferative response to Con A than to PHA (p<0.870). This situation is inverse in cord blood cells meaning that the proliferative response of CBMCs in response to Con A was less than that to PHA (p<0.104). Figure 1. The percent proliferative responses of PBMC or CBMC in response to mitogens. The mean values and the standard error of mean (SEM) of each cell group to Con A or PHA are given (* p < and ** p < 0.034). Figure 2. The correlation between the percent proliferative responses of PBMCs and CBMCs in response to Con A (A) or PHA (B). 15

4 TURK J IMMUNOL Vol.10, No.1, 2005 Figure 3. A positive correlation between proliferative effects of mitogens Con A and PHA on the PBMCs or CBMCs ( the mean proliferative response of triplicate PBMC samples from each individual person, + the mean proliferative response of triplicate CBMC samples from each individual person, the mean proliferative response of PMBC samples, the mean proliferative response of CMBC samples, the mean proliferative response of all PBMC and CBMC samples). DISCUSSION Mitogens cause mammalian cell proliferation by stimulating the mitosis 12. The mitogens Con A and PHA are specifically used for T-cell proliferation 13. Thus these mitogens are used in defining the non-specific cellular immune response. The tissue damaging seen in GVHD occurring after transplantations is known to be caused by the hyperactivation of T cells 14,15,16. To obtain a T cell reaction similar to that observed in alloantigen stimulation (as seen in transplantation or autoimmune reactions), we incubated PBMCs or CBMCs either with Con A or PHA and then determined the cell proliferative responses to them. We observed a statistically higher proliferative response by Con A stimulation when compared to that by PHA. When the differences between the control and stimulated samples were examined, the differences observed in Con A stimulated samples were significantly high. Therefore, when non-specific T cell response is questioned, as in GVHD, the use of Con A may give better results. Our results show that the proliferative response of CBMCs in response to mitogens is statistically lower than that of PBMCs. Current knowledge indicates that there are some differences between the proliferative responses of lymphocytes in response to mitogens and antigens 17,18. Mitogens stimulate T cells non-specifically 19 (without antigen processing and presentation by APCs) while activation by alloantigen involve antigen processing and presentation along with the release of certain cytokines by APCs 20. On the other hand, since the main reason underlying the tissue damage in GVHD is the "cytokine storming," the cytokine release after mitogenic stimulation 16,21 may mimic the pathologic causation of GVHD and may be used as a model in the related research 22. The lower proliferative response of CBMC in response to mitogens may be an indicative of lower capabilities of cord blood T cells to produce cytokines 23. We believe that the less intensive GUHD episodes observed in HLA matehed CBT may be due to the low cytokine production by CB T lymphocytes despite normal alloantigen presentation. The use of MTT assay in assessing the proliferative response is more advantageous when compared to other methods defining only the cell proliferation 13. However, MTT assay determines not only the cell proliferation but also the cellular activity as the formation of MTT-formazan from MTT requires mitochondrial activation. 24,25 Therefore, we believe that using MTT assay is better, as it gives more objective results. Correspondence: Faruk SÜZERGÖZ Harran Üniversitesi Fen-Edebiyat Fakültesi Biyoloji Bölümü. Þan lurfa / TURKEY suzergoz@harran.edu.tr Tel: (H) GSM: Fax:

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