1Laboratory on Thymus Research, Department of Immunology, Institute Oswaldo Cruz, The Oswaldo Cruz Foundation, Rio de Janeiro; 2Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 3Department of Neuroscience, Albert Einstein College of Medicine, University of Yeshiva, Bronx, New York 10461; and 4Necker Hospital, CNRS UMR-8603, Paris, France
Submitted 6 December 2002 ; accepted in final form 27 June 2003
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ABSTRACT |
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gap junctions; connexin43; P2 receptors; intercellular communication
Thymic epithelial cells constitute the major cellular component of the thymic microenvironment, being distributed throughout the organ, in both cortical and medullary regions. Different methods based on either ultrastructural or molecular analysis (e.g., cytokeratin expression or MHC) have indicated the existence of a heterogeneity of cortical and medullary thymic epithelial cells (19, 38, 59, 60). Cortical and medullary thymic epithelial cells (TEC) have been implicated in T cell-positive and possibly in -negative selection, respectively (4, 42). Both selection processes involve the participation of signals transmitted via T cell antigen receptors and MHC-associated peptides, as well as cytokines and adhesion molecules (3). However, the participation of other signaling molecules present in the thymic microenvironment is likely. In this context, our group has demonstrated the presence of gap junction proteins and P2 receptors in the thymic microenvironment (1, 7). Other groups have also investigated these two distinct cellular signaling mechanisms in the immune system (2, 16).
P2 receptors are extracellular nucleotide receptors classified in two major families: the P2Y (G protein-coupled receptors) and P2X (ligand-gated ion channels). In mammals, the P2Y family consists of seven receptor subtypes, namely P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13 (33, 46, 65). The P2X family consists of seven receptor subtypes (P2X1-P2X7) (46). Gap junctions are intercellular channels directly connecting the cytosol of adjacent cells, being formed by the docking of two hemichannels (connexons), each contributed by an adjacent cell. Each connexon is made of a hexamer of connexin (Cx) proteins in vertebrates and by innexins in invertebrates. There have been at least 19 isoforms of connexins cloned in rodents so far, with new members still being discovered.
With regard to the thymic epithelium, we have described functional gap junction intercellular communication in primary cultured human TEC and mouse thymic nurse epithelial cells and demonstrated the expression of Cx43 in thymic nurse epithelial cells (1). Recently, we characterized by pharmacological, functional, and molecular techniques the P2Y2 receptor in thymic nurse epithelial cells (7). Nonetheless, the physiological role of gap junctions and P2 receptors in TEC remains unclear.
Investigations in the field of calcium signaling, particularly those involving the phenomenon of intercellular propagation of calcium waves, have demonstrated that gap junction and P2 receptors constitute, in some cells, the molecular basis of two distinct mechanisms of calcium wave propagation: one dependent on a direct cytosolic cell-to-cell transmission and the other dependent on an extracellular messenger (49). Modeling and experimental data have suggested that the diffusion of an intracellular messenger, possibly Ca2+ and/or inositol 1,4,5-trisphosphate (IP3), through a gap junction-dependent mechanism, generates the intercellular wave-like calcium propagation (8, 32, 58). In some cells, however, the presence of an extracellular pathway has also been recognized, particularly involving ATP release and the paracrine activation of P2 receptors in neighboring cells (18, 51, 55).
Intercellular calcium waves (ICWs) have been detected in different primary cultured cells, including astrocytes, airway and alveolar epithelial cells, hepatocytes, retinal pigmented and lens epithelial cells, endothelial cells, and mast cells (12-15, 30, 36, 44, 50). In addition, agonist-induced ICWs have been detected in liver and hippocampal tissue slice cultures, as well as in freshly excised retinal glial cells, indicating that they are likely to occur in vivo (28, 43, 47). These studies have further suggested the possible role of ICWs in phenomena as diverse as coordination of ciliary beating in tracheal epithelial cells, hormonal regulation of hepatic function, and extraneuronal information transmission involving glial cells (14, 47, 49).
In the work described here, we have characterized for the first time the induction of ICWs in the thymic epithelium. Our data indicate the participation of both gap junctions and P2 receptors in this propagation.
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MATERIAL AND METHODS |
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Cell lines. Cortical 1-4C18 and medullary 3-10 TEC lines were kindly provided by Willem van Ewijk (Rotterdam University, The Netherlands). These cell lines were established from C57BL/6 mice, and the cortical and medullary phenotypes were determined by immunostaining with Th-3 and Th-4 antibodies, respectively (31, 41). They were further evaluated in our laboratory using a panel of anti-cytokeratin monoclonal antibodies (MAbs), which confirmed the original distinct cortical and medullary phenotypes. Cells were cultured in 10% fetal bovine serum-supplemented RPMI 1640 medium (Mediatech, Herndon, VA) at 37°C in a 5% CO2 atmosphere.
Thymic nurse epithelial cell primary culture. Thymic nurse cells (TNCs) are lymphoepithelial complexes that engulf 20-200 thymocytes. Previous studies have demonstrated that TNCs are present in subcapsular and corticomedullar junctions of the thymus (45, 63). Freshly isolated lymphoepithelial complexes gradually release the engulfed thymocytes in vitro, and after 7-10 days and a series of careful washes, it is possible to establish a thymocyte-free, fully adherent TNC primary culture. The TNC isolation was performed as previously described (35). In brief, thymi obtained from 5 to 10 BALB/c mice were minced (1mm3) with scissors and gently agitated for 20 min in RPMI 1640 medium. The released thymocytes were discarded and the thymic fragments were suspended in collagenase A solution (0.2 mg/ml; Boehringer Mannheim Biochemicals, Indianapolis, IN) and agitated at room temperature (RT) for 20 min. The supernatant was discarded and the remaining fragments were dissociated enzymatically (0.2 mg/ml collagenase A; 0.2 mg/ml dispase II, 5 µg/ml DNAse grade II; Boehringer Mannheim Biochemicals) for 20 min at 37°C. The digestion product was centrifuged and the pellet was resuspended in PBS. The cell suspension was carefully layered on top of 10 ml of fetal calf serum (GIBCO BRL, Grand Island, NY) placed in 15 ml of conical tubes. The typically heavier TNCs were allowed to sediment. TNCs obtained by this process were cultured for RNA extraction or, alternatively, plated in MatTek (Ashland, MA) imaging dishes for calcium wave experiments.
Immunofluorescence. To evaluate Cx expression, TEC were cultured on 12-mm diameter coverglasses until reaching confluence. The cells were fixed in cold (-20°C) methanol for 20 min and washed with PBS at 4°C. Cells were permeabilized and had unspecific binding sites blocked by incubation at RT with PBS containing 0.2% Triton X-100 (Sigma Chemical, St. Louis, MO) and 10% normal goat serum. The cells were then incubated overnight at 4°C with a polyclonal mouse anti-Cx 43 antibody (Zymed Laboratories, South San Francisco, CA). After being washed, the cells were incubated for 1 h with Alexa 488-conjugated secondary antibody (Molecular Probes, Eugene, OR). In some experiments, the cells were also incubated with 4',6-diamidino-2-phenylindole (DAPI, 10 µg/ml; Sigma Chemical) for 30 min for nuclear staining. The cells were analyzed under a Nikon Eclipse TE-300 microscope with phase-contrast and epifluorescence optics and photographed using a SPOT-RT digital camera (Diagnostic Instruments, Sterling Heights, MI).
RNA extraction and RT-PCR. Each 50-100 mg of tissue (heart) or confluent TEC monolayer in a 25-cm2 culture flask was dissociated in 1 ml of Trizol reagent (GIBCO BRL). The aqueous phase obtained after chloroform addition and centrifugation was transferred to new tubes and incubated at RT with 500 µl of isopropanol for 10 min to induce RNA precipitation. The isolated RNA was dissolved in diethyl-pyrocarbonate-treated water and quantified by measuring its absorbance at 260 and 280 nm.
Each RNA sample (up to 5 µg/µl) was subject to DNase I treatment and then reverse transcribed in three steps: an initial incubation at 65°C with oligo (dT)12-18 (0.05 µg/µl) and 2-deoxynucleotide 5'-triphosphate (dNTPs), followed by an incubation at 42°C with a solution containing buffer, MgCl2, DTT, and RNaseOUT. Reverse transcriptase (5 U/µl) or water (negative control) was added to each sample and incubated for 50 min at 42°C and for 15 min at 70°C. The cDNA of each sample was maintained at -70°C until the time of the experiment. PCR was performed as follows: 2 µlof the reverse transcribed product were incubated with a solution containing PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, taq DNA polymerase (0.05 U/µl), and the anti-sense and sense primers for Cx43 (sense: TACCACGCCACCACTGGC; anti-sense: AATCTCCAGGTCATCAGG) and were amplified through 30 cycles of denaturation (94°C, 30 s), annealing (55°C, 30 s), and extension (72°C, 30 s). Sizes of amplification products were determined after electrophoresis in 2% aga-rose gels.
Calcium wave detection. TEC were cultured in glass-bottomed plates (MatTek) and, after confluence, were loaded with 10 µM indo 1-AM (Molecular Probes) for 30 min at 37°C. The cells were washed three times with RPMI 1640 medium and analyzed for the presence of calcium waves in a RCM 8000 Nikon real-time confocal microscope equipped with an ultraviolet (UV) laser, using the UV small pinhole setting (54). The cells were maintained in RPMI medium, pH 7.4, at RT. Indo 1 was excited at 351 nm through a UV-transparent, high numerical aperture (n.a. 1.15) water immersion objective, and the ratio of its fluorescence intensity at two wavelengths, 390-440 and >440 nm, was monitored. After shading and background subtraction (obtained from out-of-focus planes in the same field), the indo 1 ratiometric images were continuously acquired at a frequency of 0.5 Hz. The ratiometric images before and after the mechanical stimulation were saved on an optical disk recorder as averages of 32 frames. These images were played back for measurements of calcium levels with Polygon-Star software (Nikon, Tokyo, Japan), and the average gray levels (number of pixels per area) within the regions of interest were used for analysis. Changes in Ca2+ are indicated in this manuscript as ratios, where basal Ca2+ levels correspond to ratios of 0.75 to 0.85 and maximal levels correspond to ratios of 2.3 to 2.5. In vitro calibrations with indo 1 potassium salt in EGTA or BAPTA-buffered 150 mM KCl solutions indicate that these ratios correspond to a range of 100-200 nM to >5 µM Ca2+, respectively. These Ca2+ concentrations have been verified in our quantitative Ca2+ imaging studies using dual excitation fura 2 ratiometric imaging (54).
Calcium wave induction and analysis. Mechanical stimulation was performed in one cell of the confocal field (171 x 128 µm) by gentle touching with the tip (1-2 µm diameter) of a glass micropipette as previously described (54). Calcium wave velocity was calculated considering the distance (in micrometers) between the responding cell and the mechanically stimulated cell divided by the time interval (in seconds) between the half-maximal elevation of intracellular calcium of the stimulated and responding cell.
Calcium wave amplitude was considered as the maximal relative increment of calcium of each responding cell and was calculated as a fold change by dividing the indo 1 fluorescence intensity ratio at the peak of the response by the basal indo 1 fluorescence intensity ratio before the mechanical stimulation. The efficacy of calcium wave propagation was considered as the percentage of analyzed cells in the confocal field that responded with intracellular calcium increase after the mechanical stimulation. Only cells exhibiting a >5% rise above the basal intracellular Ca2+ level were considered as responsive. In the experiments in which heptanol was applied, its stock solution (200 mM in ethanol) was prepared immediately before use.
Statistics. Control vs. treated parameters were compared by two-tail paired t-test using Microsoft Excel software. To compare the amplitude, efficacy, and velocity of ICW in all three TEC preparations, we performed the one-way ANOVA, the Tukey's test, and linear regression analysis using GraphPad Prism v. 2.0.
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RESULTS |
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Spontaneous oscillations of intracellular calcium concentration ([Ca2+]i) were frequently observed in some individual TNCs but not in cell lines (not shown). However, these oscillations did not trigger ICW.
Immediately after the mechanical stimulation, we observed an increase in intracellular calcium in the stimulated cell, which was followed by the propagation of an ICW through the neighboring cells. Such a response was observed in each of the three types of TEC preparations evaluated (Fig. 1). Ca2+ waves within individual cells were not visualized during the propagation; instead, a gradual and homogeneous increase in [Ca2+]i occurred in the cells forming the wave front before the rise of [Ca2+]i was detected in the neighboring cells (Fig. 1). Amplitude of [Ca2+]i within the stimulated cell was consistently higher than in cells to which the wave propagated. Interestingly, Ca2+ waves observed in all types of TEC cells tended to propagate either symmetrically or somewhat asymmetrically (Fig. 1, A and B).
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The kinetics of calcium increase of each responding cell was analyzed, and the general features (amplitude, velocity, and efficacy) of the ICWs propagated in each distinct TEC preparation were determined (Fig. 2). The general features of ICW in each TEC preparation were as follows: 1) TNC: velocity: 9.51 ± 1.51 µm/s, amplitude: 1.37 ± 0.12-fold the basal levels, and efficacy of spread to 82.8 ± 4.8% of the cells in the field; 2) cortical 1-4C18 cells: velocity: 6.69 ± 2.47 µm/s, amplitude: 1.70 ± 0.09-fold, and efficacy: 97.9 ± 3.1%; and 3) medullary 3-10 cells: velocity: 6.13 ± 3.68 µm/s, amplitude: 1.45 ± 0.08-fold, and efficacy: 81.4 ± 18.7%. Significant differences were observed only in the amplitude of cortical TEC Ca2+ responses, which were higher than in TNC or in the medullary TEC line (Fig. 2A). The ICW detected in all TEC preparations presented a high propagation efficacy reaching 80-100% of the cells in the confocal field (171 x 128 µm; 10-30 cells, depending on TEC population), with velocity ranging from 6 to 10 µm/s and amplitude from 1.4 to 1.7-fold the basal level.
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To elucidate the underlying mechanism of this ICW propagation, the calcium wave amplitude in each responding cell from different TEC preparations was plotted against its distance from the stimulated cell. In our analysis, propagation distances up to 100 µm were observed. As indicated in Fig. 3, A and B, the calcium wave amplitude in cortical and medullary cells remained almost unaltered during its propagation. In contrast, the calcium wave amplitude in TNC cells decreased gradually as a function of its distance of propagation (Fig. 3C). These data indicate that a regenerative mechanism dominates in Ca2+ wave propagation in 1-4C18 and 3-10 TEC lines, whereas the attenuation in TNC Ca2+ amplitude with distance suggests a more prominent diffusion component in the response.
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Gap junctions and P2 receptor dependent-calcium wave propagation. We next attempted to dissociate the contribution of gap junctions and P2 receptors to the propagation of ICWs in thymic epithelial cells. For this purpose, ICWs in different TECs were evaluated either in the presence of heptanol, a gap junction inhibitor, or in the presence of suramin, a P2 receptor antagonist (46, 48); although each of these agents affects other ion channels and receptors, such a strategy has proven to be quite effective when applied to astrocytes (54). When calcium wave propagation was evaluated in the presence of heptanol (3 mM), calcium increases of similar magnitude were detected in the mechanically stimulated cell, but calcium wave propagation to the neighboring cells was almost completely inhibited in both 1-4C18 and 3-10 cells; by contrast, a partial but significant inhibition was observed in TNCs (Fig. 4). Accordingly, in these experiments, the efficacy of calcium wave propagation induced in 1-4C18 and 3-10 cells decreased from 100.0 and 60.7 ± 27.9% to 2.1 ± 4.2 and 1.7 ± 3.7%, respectively (P < 0.001), whereas in TNCs the efficacy decreased from 88.3 ± 14.0 to 30.1 ± 29.3% (P < 0.001) (Fig. 4B). In addition, in all TEC preparations, the cells that remained responsive after heptanol treatment responded after pronounced delay and exhibited slower calcium wave propagation (Fig. 4C). In the case of 1-4C18 cells, heptanol also induced a decrease in calcium wave amplitude, whereas it did not in TNC (Fig. 4A).
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We also evaluated TECs in the presence of suramin (200 µM). Although suramin had no inhibitory effect on Ca2+ wave spread in TEC cell lines (and actually slightly but significantly increased velocity and amplitude in 3-10 cells, Fig. 5, A and C), this P2 receptor inhibitor partially inhibited the calcium wave propagation in TNCs, decreasing its efficacy from 88.3 ± 14.0 to 37.5 ± 23.6% (Fig. 5B) and its velocity from 7.81 ± 3.69 to 5.14 ± 3.19 µm/s (Fig. 5C).
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The data above indicate that gap junctions likely constitute the major mechanism of calcium wave propagation in both 1-4C18 and 3-10 TEC lines. In the case of TNCs, however, the partial blockade by both heptanol and suramin indicate that gap junctions and P2 receptors are both involved in ICW propagation.
To further test the participation of P2 receptors in ICW propagation, we pretreated TNC, 3-10, and 1-4C18 cells with UTP (100 µM) before mechanical stimulation. Each TEC preparation was mechanically stimulated in control conditions to ascertain that all cells were responsive before UTP treatment. All TEC preparations responded to UTP with an increase in [Ca2+]i (not shown), suggesting the presence of P2Y receptors in 3-10 and 1-4C18 cells, and consistent with the reported presence of P2Y2 receptors in TNCs (7). After 5 min, when the intracellular calcium returned to baseline levels, different cells in these cultures were mechanically stimulated while still in the presence of UTP. Under these conditions, TNCs exhibited significantly attenuated calcium wave propagation, lowering efficacy from 88.3 ± 14 to 20.0 ± 17%. The 3-10 and 1-4C18 TEC lines also exhibited significantly lower efficacy, being reduced 40 and
30%, respectively. These findings strengthen our conclusion that transmission of Ca2 ± waves through P2 receptors plays a more prominent role in TNC than in the TEC lines.
Cx expression in TEC. Our pharmacological data indicate the participation of gap junction and P2 receptors in TNC calcium wave propagation. In accordance, we have previously demonstrated the expression of Cx43 and functional intercellular gap junctional communication and the expression of P2Y2 receptors in TNCs (1, 7). In relation to cortical 1-4C18 and the medullary 3-10 cell lines, our data indicate that calcium wave propagation is mainly dependent on gap junctions. For this reason, the expression of Cx43 was evaluated in these cell lines. The presence of Cx43 was demonstrated by RT-PCR (Fig. 6A) and immunofluorescence (Fig. 6B) in both cell lines. We also confirmed the presence of Cx43 in TNCs, as ascertained by RTPCR (Fig. 6A). In both 1-4C18 and 3-10 cells, Cx43 protein was clearly localized in regions of cell-to-cell contact; in the case of 3-10 cells, additional prominent Cx43 staining was evident in cytoplasmic and perinuclear regions (Fig. 6B, lower panel). Gap junction functionality in these cell lines was demonstrated using lucifer yellow microinjection (data not shown), supporting the importance of gap junctions in calcium wave propagation in 1-4C18 and 3-10 cells.
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Evaluation of intercellular calcium wave transmission between TEC and thymocytes. Because the interaction between TEC and thymocytes is a fundamental event in intrathymic lymphopoiesis, we evaluated the possibility of intercellular calcium transmission between these two cells types. For this purpose, BALB/c mice-derived thymocytes were plated on top of preestablished primary TNC cells cultured as described in MATERIAL AND METHODS. These cells were cocultured for 3 h, and all cells were labeled with indo 1 and evaluated by confocal microscopy. The TNCs with adherent thymocytes were mechanically stimulated, and the intracellular calcium variations of both cell types were monitored. In these experimental conditions, the intracellular calcium increase was observed in the mechanically stimulated TNC but was not transmitted to adjacent adherent thymocytes (Fig. 7).
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DISCUSSION |
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We previously observed in different types of TEC cultures (including TNCs) that application of micromolar levels of extracellular nucleotides such as ATP and UTP elicits an increase in intracellular calcium that is related to the activation of P2Y2 receptors and mobilization of intracellular calcium stores (7). In the present work, we describe for the first time ICW in thymic epithelial cells, revealing that these cells are capable of complex calcium signaling in response to mechanical stimulation.
In various cell types, it has been demonstrated that ICW can be induced by chemical, electrical, or mechanical stimuli (28, 43, 47, 50). Mechanical stimulation has been hypothesized to initiate ICWs by activation of stretch-activated ion channels, ATP and/or UTP release by nonlytic mechanisms, and IP3 production (8, 11, 17, 26, 34, 51). Mechanical stimulation has been applied to induce ICWs in cells as diverse as retinal pigmented and lens epithelial cells (12, 30), airway and alveolar epithelial cells (36, 50), retinal glial cells (43), astrocytes (23, 29, 55), endothelial cells (15), mast cells (44), and diverse cell lines (insulinoma cells, liver epithelial cells, osteoblastic cells, and prostate cancer cells) (10, 22, 37, 51). These studies have demonstrated distinct and independent mechanisms of ICW propagation.
Gap junction-dependent, mechanically induced ICWs have been demonstrated in rabbit airway epithelial cells (27, 50), retinal pigment, and lens epithelial cells (12, 30), as well as in the ROS17/2.8 osteoblastic cell line (37). In other cell types such as mast cells (44), human polarized airway epithelial cells (34), prostate cancer, insulinoma cell lines (10, 51), WB-aB1 liver epithelial cell line (22), and mammary epithelial cell line (17), a mechanically induced intercellular calcium wave dependent on an extracellular pathway has been demonstrated, involving the release and diffusion of extracellular nucleotides and the paracrine activation of P2 receptors in the neighboring cells.
In astrocytes (18, 21, 29, 55), alveolar epithelial cells (36), the WB-F344 liver epithelial cell line, and the Cx43-transfected insulinoma cell line (10), the involvement of both gap junction and P2 receptor-dependent mechanisms has been implicated in the propagation of mechanically induced ICWs.
As regards the thymic epithelium, our studies revealed that mechanical stimulation triggers Ca2+ waves in both TEC cell lines and primary TNC cultures. As previously described, the stimulated cell was the first cell to present an increase in [Ca2+]i, being followed by responses in neighboring cells.
We evaluated two phenotypically and functionally distinct TEC lines, the 3-10 and 1-4C18 cells. The ICWs generated by these cells were similar in terms of propagation velocity and efficacy. However, the ICW in cortical 1-4C18 cells were of higher amplitude when compared with the medullary 3-10 cells. When the amplitude of calcium increase of 3-10 and 1-4C18 responding cells were plotted against the propagation distance from the stimulated cell, we observed that the amplitude of the calcium elevation in 1-4C18 and 3-10 responding cells remained almost unaltered, indicating the possibility that a regenerative mechanism may underlie calcium wave propagation in these cells. Interestingly, the ICWs generated in 3-10 and 1-4C18 cells were almost completely blocked by heptanol treatment, whereas suramin did not induce any inhibitory effect. These data strongly suggest that in both cortical and medullary TEC lines, ICW propagation is mediated primarily by a gap junction-dependent mechanism.
To ascertain that the ICWs were not restricted to immortalized TEC lines, we further demonstrated the same phenomenon in TNC primary cultures, which are believed to have a subcapsular and corticomedullary localization within the thymus (45).
In our experiments, both heptanol and suramin partially inhibited the intercellular calcium waves in TNCs, indicating the involvement of both gap junction and P2 receptor-dependent pathways in ICW propagation. The desensitization of P2 receptors by previous UTP application inhibited 80% of the mechanically induced ICWs in TNCs, thus corroborating the participation of P2 receptors in ICW propagation.
Our results suggest the involvement of gap junctions and P2 receptors in the propagation of calcium waves in TNCs and the involvement of gap junctions in calcium wave propagation in TEC lines. Interestingly, preliminary evaluation of another TEC line, the IT-76M1 cells, which exhibit a mixed cortical/medullary phenotype (64), indicates the participation of both P2 receptors and gap junctions in ICW propagation (data not shown), suggesting that the participation of P2 receptors in ICW propagation is not limited to primary cultured TEC.
When the transmission of ICW was investigated between TNCs and thymocytes, the mechanically stimulated TNC responded with an increase in intracellular calcium, but the calcium wave did not propagate to neighboring adherent thymocytes. These data suggest the possibility of communication compartments within the thymus, where calcium waves may preferentially provide signaling among cells of a given type. However, the investigation of ICW transmission between TNC and thymocytes in freshly isolated three-dimensional lymphoepithelial complexes, as well as thymic slices, are necessary to better establish such a concept.
Thymic epithelial network forms a three-dimensional spongelike stroma with numerous TEC-to-TEC contacts (61). In addition, Cx43 expression has been demonstrated in TEC network in both cortical and medullary regions of thymus sections (1). These data indicate the existence in thymus of the anatomical and molecular basis for ICW propagation.
In different tissues and cells, ICW has been directly triggered by soluble factors. Some examples are vasopressin in liver, glutamate in astrocytes, and ATP in freshly excised retinal glial cells (14, 43, 47). Our data indicate that gap junctions and P2 receptors are important components in ICW transmission among TECs. Thus, although not evaluated in the present work, ATP and UTP constitute important potential candidates as soluble initiators of ICW in thymic epithelium. Two facts support this possibility: the expression of different P2 receptors in thymic epithelium, i.e., P2Y2, P2X2, P2X3, P2X6, and P2X7 (7, 24), and the presence of noradrenergic innervation in thymic septa, subcapsular region, cortex, and corticomedullary junction (20, 40), which potentially may also corelease nucleotides (57). Such a P2 receptor-triggered ICW might potentially affect synthesis of interleukin-6 and prostaglandin E2 because such modulation has been observed in TEC after ATP treatment (39, 62).
Because the thymus physiology is influenced by classic hormones, neuropeptides, and neurotransmitters, some of these molecules, mainly those which stimulate IP3, also constitute potential candidates as soluble stimuli for ICW triggering in thymic epithelium (53). Future investigations using ex vivo thymic preparations may clarify the existence of ICW in physiological conditions and the importance of neuroendocrine products in its initiation.
In conclusion, the propagation of ICW in the thymic epithelium described here reveals that these cells are capable of responding with a complex intercellular signaling and also suggests that ICW may require functional gap junctions and P2 receptors in these cells.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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