Stem Cells, Vol. 15, No. 3, 229-236,
May 1997
© 1997 AlphaMed Press
Adhesion of Thymocytes to Bone Marrow Stromal Cells: Regulation by bFGF and IFN-
Mira Barda-Saada,
An-Sheng Zhanga,
Dov Ziporib,
Leon A. Rozenszajna
a Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel;
b Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel
Key Words. T cell-bone marrow stroma cell adhesion • Microenvironment • Fluorimetric cell quantitation • Calcein-AM • FDA • bFGF • IFN-
Dr. Dov Zipori, Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.
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Abstract
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We recently reported on selective interactions between immature T cell subpopulations and bone marrow (BM) stromal cells. To further study this process, we first examined the efficacy of methods estimating cell-cell adhesion and then investigated the effects of cytokines on thymocyte-stroma associations. Techniques based on the use of the fluorochromes calcein-acetomethylester (calcein-AM) and fluorescein diacetate (FDA) were studied and compared to regular cell counting methods. With calcein-AM labeling, the retention time was relatively long, while with FDA labeling, there was a rapid cellular efflux. Using calcein-AM, we developed an accurate quantitative fluorometric assay for determining the adherence of thymocytes to a BM stromal cell line (MBA-13). A maximal fraction of about 29% thymocytes was found to adhere to confluent MBA-13 cell layers after four to six h of coculture. Whereas interleukin 1 did not change the rate of adhesion of thymocytes to the stroma, interferon- (IFN-) significantly increased adhesion. Basic fibroblast growth factor (bFGF) had a dose-dependent biphasic effect on thymocyte adhesion, and a greater fraction of double negative thymocytes adhered to stroma pretreated with bFGF. Taken together, these results suggest that IFN- and bFGF modulate T cells-BM stromal cell adhesion.
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Introduction
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Accumulating evidence suggests that bone marrow (BM) stromal cells create a lymphohemopoietic microenvironment that regulates proliferation and differentiation of stem cells. This process requires direct cell-cell interactions between stroma cells and stem cells. In addition, cytokines and extracellular matrix components contribute to the regulatory mechanism [1-5]. Our previous studies showed that the BM contains T cell colony forming units [6] and that BM stromal cells are able to maintain thymocytes in long-term cultures [7]. During the incubation period, a gradual increase in the incidence of immature cells occurred in these cultures. Recently we established a culture system in which murine immature thymocyte populations selectively adhere to BM stromal cells [8]. We used this in vitro system as a model to study T cell-BM stromal cell interactions.
To obtain a comprehensive understanding of the interactions between T cell precursors and BM stromal cells, a reliable quantitative assay for cell-cell adhesion is required. Fluorometric assays are superior to the more widely used radioisotopic methods, due to their higher sensitivity and safer handling [9]. Several fluorochromes including calcein-acetomethylester (calcein-AM), CFDA, CFSE, BCECF-AM, DiI and fluorescein diacetate (FDA) have been investigated [9-13]. A low spontaneous release rate is an important criterion in the search for a suitable fluorochrome. Previous studies showed that thymocytes could be stained efficiently and homogeneously by both calcein-AM and FDA [12, 13]. Calcein-AM was regarded as the best candidate for cellular adhesion assays because of its high labeling efficiency, long retention time, lack of influence on cellular functions, independence of fluorescence on pH and absence of fluorescence transfer to unlabeled cells. Compared to cytotoxicity experiments, the study of interactions between thymocytes and BM stromal cells requires prolonged incubation periods [8, 14]. In the present investigation, we show that calcein-AM fulfills the requirements of this assay. Stromal cells have been shown to be affected by a variety of cytokines. The addition of interleukin 1 (IL-1) to cultured thymic lobes induced proliferation and differentiation of double negative (CD4–CD8–) precursor cells to functionally competent T cells [15, 16]. This cytokine was further shown to upregulate the expression of vascular cell adhesion molecule-1 on BM derived stromal cells [17]. Similarly, interferon- (IFN-) upregulated the expression of VLA-5 and VLA-6 on thymic-derived stromal cells and consequently increased thymocyte binding [18]. We therefore chose to study these cytokines along with basic fibroblast growth factor (bFGF) which has been shown to directly affect purified hematopoietic stem cells and to act indirectly by modulating stroma cell function [19, 20].
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Materials and Methods
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Thymocyte Labeling
BALB/c mice (male, 6 -10 weeks) served as donors of thymocytes. Calcein-AM (MW: 994.87; Molecular Probes; Eugene, OR) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mM, and stored at -20°C. The labeling was performed, as previously described [10], with minor modifications. Thymocytes were suspended in Dulbecco's modified Eagle's medium (DMEM), and supplemented with 10% fetal calf serum (FCS), 107 cells/ml. Four milliliters of cell suspension were then mixed with 20 ml of calcein-AM stock solution (final concentration, 5 mM). After 15 min incubation in a water bath at 37°C, the labeling was stopped by the addition of cold (4°C) DMEM containing 10% FCS. The cells were washed twice and centrifuged at 4°C. The cell pellet was resuspended in phosphate-buffered saline (PBS) and viability was determined by trypan blue dye exclusion. FDA (MW: 416.4; Sigma; St. Louis, MO) was dissolved in acetone at 5 mg/ml. Thymocyte staining with FDA was performed according to the method described by Maeda et al. [12], with minor modifications. Thymocytes were suspended in RPMI 1640, supplemented with 10% FCS, 107 cells/ml. Nine milliliters of cell suspension were mixed with 1 ml of FDA stock solution and incubated at room temperature for 15 min. The cells were then washed twice, centrifuged at 4°C, and resuspended in PBS. Viability was determined by trypan blue dye exclusion.
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Determination of Fluorescence Intensity and Retention of Cellular Fluorescence
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The labeled thymocytes were diluted with PBS. OD values were measured, using a fluorescence spectrophotometer RF-5000 (Shimadzu; Kyoto, Japan), excitation wave lengths 485 nm and 488 nm from a Xenon lamp, and emission wave lengths 530 nm and 517 nm for the thymocytes labeled with calcein-AM and FDA, respectively. A 5 nm band width was adopted. The cellular fluorescence retention time was assessed by suspending the labeled thymocytes -106 cells/ml in DMEM supplemented with 10% FCS, 2 mM glutamine, and 5 x 10–5 M 2-mercaptoethanol, and by incubating them at 37°C in a humidified atmosphere of 10% CO2 in air. After 0.5, 1, 2, 4, 6 and 24 h of incubation, the thymocytes were separated from the liquid phase by centrifugation. The supernatant was carefully collected, and the cell pellet resuspended in PBS. The cellular fluorescence retention was calculated from the OD values in the liquid phase and from the fluorescence that remained in the cell fraction. Cellular viability was evaluated by trypan blue exclusion.
The samples were tested for possible spontaneous changes of OD measuring the OD before and after 24 h and 48 h of incubation, at 4°C and at 37°C, with and without exposure to light. No changes were observed, indicating that the fluorescence did not decay and that it was insensitive to light.
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Quantitation of Thymocyte Adhesion To Stromal Cells by Cell Counting and Fluorometric Assay
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Adherent assay of T cells to stromal cell lines was performed as described [8]. MBA-13 stromal cell line has been derived from nude/ICR mouse bone marrow [21-23]. These anchorage-dependent cells were grown to confluence and maintained at this state for one week culture in DMEM, supplemented with 10% FCS (Bio-Lab. Ltd.; Jerusalem, Israel), at 37°C with 10% CO2 in air. Unlabeled or labeled thymocytes were suspended in culture medium identical to that used for the fluorescence retention test, 106 cells/ml, and were seeded onto the confluent stroma layers. After the periods of coculture specified above, the nonadherent and adherent thymocytes were collected separately.
The adhesion of thymocytes was determined as follows: The nonadherent cells were harvested by gentle washing. In order to collect the adherent thymocytes, both the stromal cells and the thymocytes adhering to the stroma were first detached with 0.5% trypsin and 0.02% EDTA in PBS for 5 min and then washed once and centrifuged. The cell pellet was resuspended and diluted in DMEM supplemented with 10% FCS, seeded in an empty culture dish and incubated for an additional hour to allow the stromal cells to adhere. The thymocytes in the liquid phase were then collected. The adhesion of thymocytes was evaluated both by a cell counting method and by fluorometric assay.
The nonadherent and adherent thymocytes were evaluated in a hemocytometer for trypan blue exclusion. The adhesion was calculated as follows:
| (1) |
By fluorimetric assay: The adhesion of labeled thymocytes was evaluated by direct measurement of fluorescence intensity of nonadherent thymocyte population, which was collected from the liquid phase of the cultures, versus the adherent population which was detached by trypsin treatment. The fluorescence of stromal cells, incubated without thymocytes and treated as above, served as a control and was taken into consideration in calculating fluorescence values.
The adhesion of labeled thymocytes evaluated by fluorescence intensity (FI) measurement was calculated according to the following formula wherein:
| (2) |
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Effect of bFGF, IFN- and il-1 on T Cell-Stromal Cell Interaction
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To assess the effect of bFGF, MBA-13 cells were seeded at 1 x 104/ml in the presence or absence of bFGF (0-4,000 ng/ml). After 10-14 days, confluent cultures were washed to remove residual bFGF, the thymocytes were seeded (1 x 106 cells/ml), the cultures were incubated at 37°C in a humidified atmosphere of 10% CO2 for an additional 24 h, and cell-cell adhesion was measured as described above. Adhesion molecule expression: CD11a, CD2 and CD49d of adherent and nonadherent thymocytes was analyzed by using flow cytometry. IFN- (0-500 U/ml) or IL-1 (0-5 U/ml) was added to a culture containing MBA-13 confluent culture and thymocytes (1 x 106 cells/ml). Cell-cell adhesion was quantified after 24 h of culture.
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Flow Cytometry
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Thymocytes were washed with PBS containing 0.02% sodium azid and incubated for 30 min at 4°C with the relevant antibodies. After washing, the cells were analyzed for two-color fluorescence with a fluorescence-activated cell sorter ([FACScan] Becton Dickinson; San Jose, CA) with logarithmic intensity scales. About 5 x 103 cells were scored using Lysis II software (Becton Dickinson). The following monoclonal antibodies (mAb) were used: fluorescein isothiocyanate-conjugated anti-mouse CD8 and phycoerythrin-conjugated anti-mouse CD4. The expression of adhesion molecules was analyzed by using specific mAb: rat anti-mouse CD11a, antimouse CD2 and anti-mouse CD49d (Pharmingen; San Diego, CA).
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Scanning Electron Microscope Analysis
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The morphology of calcein-AM labeled thymocytes and their interaction with MBA-13 stromal cells were analyzed with a scanning electron microscope (Jeol SEM 840; Tokyo, Japan) and compared with those of unlabeled fresh thymocytes. For the morphological examination, a single cell suspension was washed twice with PBS without Ca2+ and Mg2+, and fixed with 2.5% glutaraldehyde in PBS for 1 h at room temperature and 2 h at 4°C. The thymocytes were then washed twice with PBS. For adhesion analysis, thymocytes were seeded in a 24-well plate onto round, micro-cover-glasses covered with a confluent layer of MBA-13 stromal cells. After 6 h of coculture, the nonadherent cells were gently removed. The thymocytes adhering to the stroma were fixed as above. All the samples were then dehydrated, critical point-dried, coated with gold by vacuum evaporation, and stored till examined.
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Results
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Evaluation of Thymocyte Adhesion to Stromal Cells
Thymocytes labeled either with calcein-AM or FDA were serially diluted in PBS, and their FI measured. The results with both fluorochromes showed a similar linear correlation, with a minimal detection limit of <103 cells/ml ( Fig. 1A). A trypan blue exclusion test, before and after labeling, showed that the percentage of viable cells after labeling was >99%. Figure 1B illustrates the retention of cellular fluorescence by thymocytes labeled with calcein-AM or with FDA during a 24 h period of incubation. FDA has a rapid cellular efflux. After 1 h of incubation, almost all the FDA was released into the liquid phase, while less than 5% remained within the cells. Calcein-AM was retained by the cellular membrane with little leakage. After 6 h of incubation, approximately 85% of labeling still remained within the cells. These results indicate that calcein-AM is a more suitable fluorochrome for thymocyte labeling than FDA.
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Figure 1. Fluorometric assay of cell-cell adhesion. (A) Correlation between concentration of thymocytes stained by calcein-AM or FDA and fluorescence intensity (OD). Results from one typical experiment are shown. (B) Time-dependent cellular fluorescence retention properties of calcein-AM or FDA. Results from three separate experiments (mean ± SD) are presented. (C) Time-dependent thymocyte adhesion to MBA-13 stromal cells. Results from three separate experiments (mean ± SD) were summarized. Student's t-test analysis showed no significant differences between the unlabeled and labeled thymocytes, p < 0.05.
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Our previous study indicated that the MBA-13 stromal cell line has a selective ability to bind immature thymocytes [8]. In this study, we investigated this phenomenon in greater detail by coculturing unlabeled thymocytes with confluent stromal cell layers and evaluating the numbers of adherent thymocytes as a function of incubation time ( Fig. 1C). An adhesion plateau of about 29% was reached after 4-6 h, and this percentage remained constant during the ensuing 20 h suggesting that 4-6 h of coculture are sufficient for evaluating the interactions between MBA-13 stromal cells and thymocytes. Similar adhesion profiles were obtained with unlabeled and labeled thymocytes, either by direct OD measurements or by cell counts ( Fig. 1C). No morphological changes of calcein-AM labeled thymocytes, and no alteration of the interaction between the calcein-AM labeled cells and stromal cells, could be detected by scanning electron microscope analysis ( Fig. 2). These results indicated that calcein-AM labeling did not affect the adhesive properties of thymocytes. Thus, the rapid fluorometric measurements may replace the cell counting method. Furthermore, cell counting and the use of fluorochromes yielded identical results ( Fig. 1), indicating they are similarly reliable.
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Effect of Cytokines on Thymocyte Adhesion To BM Stromal Cells
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We have recently reported the effect of bFGF on stromal cell phenotype and hemopoietic functions [20]. This cytokine was found to be stimulatory to stroma-dependent myelopoiesis in pg/ml, and highly inhibitory in ng/ml concentrations. As shown ( Fig. 3A), a biphasic effect was monitored. At 0.1-10 ng/ml, bFGF caused increased adhesion of thymocytes to the stroma. Higher concentrations were ineffective, and only at mg/ml concentrations an additional peak of adhesion could be seen. IFN- had an adhesion-inducing effect which was similar in magnitude to that obtained with bFGF ( Fig. 3B). By contrast, IL-1 did not have a significant effect on thymocyte adhesion to the stroma ( Fig. 3C). To determine whether the increased adhesion was due to selective binding of a specific T cell subpopulation, we analyzed the phenotype of the cells adherent to the stroma, as well as that of the nonadherent cells. As shown in Figure 4, bFGF at the higher concentrations used seems to increase mainly the adhesion of double negative cells (CD4–CD8–). Low expression of CD11a molecule was found on the adherent thymocytes collected from these cultures ( Table 1).
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Figure 3. Effect of cytokines on thymocyte adherence to BM stromal cells. Thymocytes were cultured for 24 h over confluent layers of MBA-13 cells pretreated with bFGF (0-4,000 ng/ml) (A), or in the presence of IFN- (4 x 105 U/ml) (B) or IL-1 (0-5 U/ml) (C). The number of the adherent and the nonadherent cells was determined and the adherence percentage was calculated. The figures represent the mean ± SE Student's t-test analysis showed significant differences: *a: p < 0.021; *b: p < 0.046; *c: p < 0.015.
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Figure 4. Adhesion of T cell populations to bFGF-treated stromal cells. Thymocytes were cultured for 24 h over confluent layers of bFGF-treated (0-2,000 ng/ml) stromal cells. The number of adherent and nonadherent thymocytes was determined and their surface phenotype was analyzed for CD4 and CD8 antigen expression. Thymocytes were stained with the relevant mAbs and analyzed for two-color fluorescence with a FACScan (Becton Dickinson). About 5 x 103 cells were scored using Lysis II software (Becton Dickinson). The data represent the mean ± SE Student's t-test analysis showed a significant difference in the number of double negative adherent thymocytes between 0 and 2,000 ng/ml, p < 0.047.
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Discussion
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It is well known that bone marrow progenitor cells seed the thymus where they proliferate and differentiate into mature T lymphocytes during the processes of positive and negative selection [24]. Recently accumulated evidence demonstrates the involvement of the BM in T lymphopoiesis and the central role of stromal cells in the process [8, 25-28]. While considerable progress has been made in isolation and characterization of hematopoietic stem cells, little is known about the properties of stroma cells that regulate lymphohemopoiesis [29]. We have shown that stromal cells from the bone marrow support myelopoiesis, pre-B lymphopoiesis and precursor T cell proliferation [5, 7, 30]. Mesenchymal stromal cell lines of thymic origin shared these properties. It is thus possible that similar mesenchymal stromal populations exist in both the BM and the thymus and provide signals for T cell growth or differentiation. In the present study, we utilized a BM stromal cell line to investigate one aspect of thymic T cell function, i.e., adhesion capabilities.
We compared available assays for the quantitation of cell adhesion. The fluorometric assay described here was compared to actual cell counting and was found to produce similar results.
Both calcein-AM and FDA are nonfluorescent compounds and can be converted in viable cells intracellularly into green fluorescent derivatives by cytoplasmic nonspecific esterases [11, 12, 14]. The time-dependent retention of calcein-AM and FDA in labeled thymocytes was studied. The former had a low spontaneous release rate—about 96% and 85% of fluorescence remaining within the thymocytes after 2 h and 6 h of incubation, respectively ( Fig. 1). Fluorescent FDA was observed to be poorly retained by labeled thymocytes, with only 5% of fluorescence remaining after 1 h incubation. Despite the fact that calcein-AM and FDA shared high staining efficiency and no toxic effects, our results indicate that calcein-AM is superior to FDA because of its high retention rate. Scanning electron microscope analysis supported the credibility of the assay by showing that the labeled thymocytes adhered to the stroma without any evident signs of cell damage ( Fig. 2).
The cytokines IL-1, IFN- and bFGF were tested to study their effects on T cell-stroma interaction. IL-1 is known to induce proliferation of preactivated cells [31]. bFGF affects hemopoiesis by its ability to induce proliferation and differentiation of early hemopoietic cells [19, 32], and it may also act indirectly by modulating stroma cell function [20]. IFN- has, in vitro, an inhibitory action on hematopoiesis [33]. Our previous studies indicated that stromal functions are altered by bFGF [20]. Here we show that under the same experimental conditions, i.e., upon preincubation of stroma with bFGF, modified adhesion of thymocytes to the stroma occur. IFN- was found to have an effect on adhesion molecule expression within 4-24 h. We therefore examined its effect shortly following addition to thymocyte stroma coculture. This indeed resulted in increased adhesion. By contrast, an additional cytokine, IL-1, that also has an immediate effect on expression of adhesion molecules did not modify the adhesion of thymocyte to stroma ( Fig. 3).
The biphasic effect of bFGF seems to stem from selective adhesion of T cell subpopulations. An increase in the number of adherent double negative cells occurred at mg/ml concentrations of bFGF with a low expression of CD11a molecule ( Table 1). Since the increase in the number of adherent cells was not accompanied by reduction in the nonadherent fraction, it is probable that T cell proliferation occurred during the 24 h incubation period. The increase in adherent cells at pg/ml to ng/ml concentrations appears to involve, also, the adhesion of CD4+ cells ( Fig. 4). Single positive cells do not significantly adhere to unstimulated stroma [8]. Thus, the increased adhesion of CD4+ cells following stimulation with bFGF may be due to activation of adhesion molecules on either the stroma or the T cells. Whether the accumulation of double negative cells in the nonadherent fraction is due to a direct effect of bFGF or to indirect activation of cytokine secretion remains to be seen.
In conclusion, immature thymocytes preferentially bind to the BM stromal cell line MBA-13. We have described here an accurate quantitative fluorometric assay using calcein-AM for determining T cell binding which could replace the conventional time-consuming cell counting method.
The adhesion capability was found to be regulated by IFN- and bFGF. The latter elicited a biphasic response: in relatively high concentrations it increased adhesion of double negative cells, while lower concentrations induced the adhesion of CD4+ cells.
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Acknowledgments
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Dov Zipori is an incumbent of the Joe and Celia Weinstein Professorial Chair at the Weizmann Institute of Science. We thank Dr. Shifra Hochberg and Ms. H. Deshen, M.Sc., for editorial assistance.
The research was supported in part by the Shapiro Cancer Research Fund, Bar-Ilan University, Ramat-Gan, Israel.
Mira Barda-Saad is a Ph.D. student, and this study is part of her research project.
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accepted for publication January 8, 1997.
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Abstract
Introduction
