ARTICLE |
Correspondence to: Sebastiano Miscia, Istituto di Morfologia Umana Normale, Università degli Studi G. D’Annunzio, Via dei Vestini, 6, 66100 Chieti, Italy.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The response of T-cells to peptide antigen plus major histocompatibility complex (MHC) consists of a series of cellular events collectively called T-cell activation. An essential component of this pathway is phospholipase C (PLC)1, whose hydrolytic activity increases rapidly after binding of ligands to the T-cell receptor (TCR) and consequent activation of tyrosine kinases. Recent studies also suggest a GTP binding protein-dependent activation of PLCß during the early steps of T-cell activation. On the basis of these findings, we first checked the expression of PLC isoforms by Western blotting and by confocal and electron microscopy techniques, and then we looked for the phosphoinositide breakdown induced by CD3 engagement in cord and adult T-lymphocytes. Our results indicated that PLCß1 was almost exclusively expressed in cord T-cells, whereas PLCß2 was more strongly represented in the adult. The amount of PLC
1 was found to be larger in the adult than in cord cells. No significant differences were found in PLC
2 and
2 expression. PLC
1 was scarcely detectable. On CD3 stimulation, adult lymphocytes gave rise, as expected, to a dramatic increase in phosphoinositide breakdown, whereas in cord cells this response was scarcely detected. These results indicate that a shift in PLC expression occurs in the postnatal period and that this change is associated with induction of the capability to respond to CD3 engagement with phosphoinositide hydrolysis. (J Histochem Cytochem 47:929935, 1999)
Key Words: PLC, T-lymphocytes, cord cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell activation induced by the engagement of the T-cell receptor (TCR) is mediated by signals that are transduced from the plasma membrane to the nucleus of the cell. TCR stimulation activates protein tyrosine kinases (PTKs) (1, leading to an increased cytoplasmic level of inositol phosphates and calcium concentration (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
T-lymphocyte Isolation
Mononuclear fractions were prepared from umbilical cord blood and from peripheral blood of women immediately before delivery. For positive isolation of CD2+ cells, mononuclear cells were incubated with magnetic beads coated with an anti-CD2 MAb (Dynal; Oslo, Norway) for 30 min at 4C with gentle rotation. Isolated CD2+ cells were recovered by means of a magnet and used for immunoprecipitation of PLC isoforms. Alternatively, mononuclear fractions were depleted first from adherent cells by incubating samples in plastic dishes overnight and then from B-lymphocytes using magnetic beads coated with an anti-CD19 MAb (Dynal). The cell suspensions were then used for the immunocytochemical analysis of PLC expression or for the inositol lipid metabolism assay.
PLC Isoform Immunoprecipitation
CD2+ cells (50 million), washed in PBS, were resuspended in lysis buffer [10 mM Tris-HCl buffer, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mg/ml bovine serum albumin (BSA), 1 mM vanadate, 50 mM sodium fluoride] and left on ice for 30 min. After determination of protein concentration, samples were normalized at 400 µg protein. Lysates were incubated at 4C for 60 min with 1 µg of anti-PLC ß1, ß2, 1,
2,
1, or
2 polyclonal antibodies (Santa Cruz Biotechnology; Santa Cruz, CA), previously coupled to goat anti-rabbit IgG magnetic beads. Immunocomplexes were collected by a magnet and washed several times with RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) in the presence of protease inhibitors.
Immunoblot Analysis
PLC immunoprecipitates were resuspended in SDS sample buffer, electrophoresed in 8% SDS-PAGE, transferred onto nitrocellulose membranes, and incubated for 1 hr at room temperature (RT) with the respective anti-PLC isoform antibody (Santa Cruz Biotechnology; 1 µg/ml) in wash buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween-20), 5% nonfat milk. Immunoreactive bands were detected by the ECL system (Amersham; Milan, Italy) using a peroxidase-conjugated goat anti-rabbit IgG antibody (Biorad Laboratories; Hercules, CA) 1:3000 in wash buffer, 5% nonfat milk. Internal controls, obtained by incubating membranes only with secondary antibody, always yielded negative results.
Densitometric Analysis
Densitometric analyses were carried out by means of a Quantimet 500 Plus (Leica; Cambridge, UK) to determine the gray intensity levels, using ISO (transmission density standard Kodak 152-3406; Kodak, Rochester NY).
Confocal Analysis of PLC Isoform Expression
Macrophages and B-depleted samples were fixed in formaldehyde 3.7% and permeabilized with PBS 0.1% Triton X-100. Slides were then incubated with anti-PLC ß1, ß2, 1,
2,
1, and
2 antibodies (1:100; Santa Cruz Biotechnology) and reacted with fluorescein (FITC)-conjugated anti-rabbit IgG antibody (1:100; Molecular Probes, Eugene, OR). To avoid unspecific binding, all antibody solutions were prepared in PBS, 4 mg/ml NGS, 4 mg/ml human IgG. Samples were then mounted in glycerol containing 1 µg/ml propidium iodide (PI) to counterstain nuclei. To check the experimental procedures, slides were incubated only with the secondary antibody, and did not show any FITC labeling. Confocal analysis was carried out with a TCS 4D (Leica; Heidelberg, Germany). To compare the expression of the isozymes in cord and adult blood samples, the laser beam output energy, the detector voltage, and the pinhole in front of the photomultiplier, differently set for FITC and PI acquisition, were maintained constant during the observation. The serial optical sections obtained by excitation of FITC and PI were finally merged and analyzed by a 3D processing system.
Immunoelectron Microscopy
T-cells were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer for 1 hr at 4C. Pellets were dehydrated in dimethylformamide and embedded in Lowicryl K4M, followed by UV polymerization. To block nonspecific binding sites, grids were treated with a blocking buffer (PBS, 0.1% Tween, 0.1% BSA, 1% nonfat milk, 3% NGS), pH 7.6 for 30 min at RT. Sections were incubated with the anti-PLC isoforms ß1, ß2, 1, and
2 diluted 1:5 in blocking buffer for 2 hr, followed by a secondary antibody conjugated with 20-nm colloidal gold particles (BioCell, Cardiff, UK; 1:5 in blocking buffer, pH 8.2). Grids were stained with uranyl acetate and examined with a Zeiss 109 electron microscope at 80 kV. Grids incubated only with the secondary antibody represented the negative control of the experimental procedure and did not display any reactivity.
Phosphoinositide Hydrolysis
Macrophage- and B-lymphocyte-depleted samples were incubated with [3H]-myoinositol (35 µCi/ml, 1020 Ci/mmol; Amersham) for 2 hr at 37C in RPMI 1640 50% autologous serum. The cells were rinsed twice with HEPES-buffered RPMI 1640 containing 20 mM LiCl and 1 mg/ml BSA and incubated in the same solution at 37C for 15 min. Samples were then incubated with anti-CD3 antibody (Sigma; Milan, Italy) or with isotype control antibody (10 µg/107 cells). The antibody was allowed to bind for 30 min on ice and stimulation was initiated by incubation in a 37C water bath for 2 min. Immediately after this incubation, 5 ml of ice-cold PBS was added and cells were collected by centrifugation at 4C. After washes in PBS, pellets were treated with 0.6 ml of ice-cold 7.5% perchloric acid. Cell debris was pelletted by centrifugation and supernatants were diluted 1:15 with 30 mM ammonium formate/2 mM Na-tetraborate and applied to an AG1-X8 ion exchange column (Biorad Laboratories). The elution of the inositol phosphate esters from the column was performed by stepwise addition of 60 mM ammonium formate/5 mM sodium tetraborate (for glycerophospho[3H]-inositol); 0.2 M ammonium formate/0.1 M formic acid (for [3H]-InsP); 0.4 M ammonium formate/0.1 M formic acid (for [3H]InsP2); 0.8 M ammonium formate/0.1 M formic acid (for [3H]-InsP3); 1.2 M ammonium formate/0.1 M formic acid (for [3H]InsP4). Eluted fractions were then analyzed by ß-scintillation counting.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoprecipitation of the different PLC isoforms from equal amounts of cord and adult T-lymphocyte protein was performed and each immunoprecipitated isoform was detected by Western blotting. As shown in Figure 1, in adult T-lymphocytes PLCß2 and PLC1 are strongly represented, whereas the reaction in cord cells displayed a lower expression of such isozymes. PLCß1 immunoreaction was higher in cord T-cells. No critical differences in PLC
2 expression were detected between cord and adult T-lymphocytes. PLC
1 was weakly expressed and slightly higher in the cord. The analysis of PLC
2 expression did not indicate differences between cord and adult blood T-cells. The densitometric analysis (sum of all gray values obtained from each pixel inside the selected bands) of the Western blots is reported in Table 1 as the mean of five different experiments. Significance of results was determined by Student's t-test. We next sought to determine the intracellular distribution of the PLC isoforms by confocal microscopy. Macrophage- and B-lymphocyte depleted samples were analyzed and the images confirmed the western blot results showing that essentially all the isoforms were located at the cytoplasmic level (green fluorescence), although inside the nucleus (red fluorescence) some light-green granular reaction was also evident to a lower extent (Figure 2). PLCß1 was mainly represented in cord cells, where green cytoplasmic immunostaining was detected, whereas adult T-lymphocytes were only poorly stained (Figure 2A and Figure 2B). PLCß2 immunocytochemical analysis, on the other hand, showed a brilliant and homogeneous fluorescence in the cytoplasm of the adult cells, accompanied by distinct evidence of clumped or finely dispersed spots of fluorescence in the nuclear compartment (Figure 2C). A lighter reactivity was detected in cord cells, both at the cytoplasmic and the nuclear level (Figure 2D). Bright PLC
1 immunofluorescence was observed in both adult and cord T-lymphocytes, appearing slightly lower in cord (Figure 2E and Figure 2F). No significant differences were detected when lymphocytes were stained for
2 (Figure 2G and Figure 2H) and
2 (Figure 2I and Figure 2L) expression. The scarce recovery of green fluorescence, together with its rapid fading by the laser beam, did not enable us to acquire the images and to evaluate the cellular distribution of the
1-isoform. To assess the specific subcellular distribution of the PLC isoforms, we performed an immunoelectron microscopic analysis on both cord and adult CD2+ cells. Because the expression of PLC
1 was very weak in both groups and no differential expression of PLC
2 was observed, we focused our attention on PLCß and
. To preserve as well as possible the antigenic properties of these molecules, we embedded the samples in Lowicryl K4M, a resin that is known to minimize reactivity with the antigen (
1 in adult cells revealed features that differed from those of the other isoforms. Distinct clusters of gold particles scattered throughout fine, dispersed labeling were typically detected (Figure 3E). A clustered organization was also found in cord cells, although the basal diffuse labeling was less evident (Figure 3F). This was in line with the finding of a lower expression in these cells by both Western blot and immunofluorescence analyses. The clustered expression and the topographic arrangement of gold granules related to the PLC
2 were similar in the cord and adult blood samples (Figure 3G and Figure 3H). Of interest is that grains of PLC
2 strictly associated with mitochondria were frequently observed.
|
|
|
|
The finding of a differential expression of the PLC isoforms between cord and adult T-cells prompted us to investigate the activity of PLC by measuring phosphoinositide hydrolysis after stimulation of intact cells with anti-CD3. To assess the specificity of the response, adult and cord T-lymphocytes were stimulated with an isotype-matched control antibody. The results (Table 2) clearly indicated that anti-CD3 stimulation of adult T-cells gave rise to high levels of inositol phosphates, whereas in cord T-lymphocytes the production of inositol phosphates was scarcely detectable. Stimulation of both types of T-cells with the isotype-matched control antibody failed to produce any response.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phospholipase C plays a key role in the signal transduction pathway for several hormones and growth factors. It is responsible for the hydrolysis of phosphatidylinositol-bis-phosphate, thus producing two critical intracellular second messenger molecules, diacylglycerol (DAG) and inositol 3,4,5 trisphosphate (IP3). The PLC isozymes can be divided into three types: ß, , and
. Because PLCß and PLC
1 are known to play a pivotal role in the cascade of molecular events generated by T-cell receptor stimulation (
1 levels, and similar expression of PLC
2 and
2. It should be emphasized that the different distribution of the PLC isoforms is not related to the relative representation of CD4 and CD8 subsets or of natural killer (NK) cells, because the ratio of CD4 and CD8 cells and the proportion of NK cells are invariable in cord and adult blood (
1 in cord T-cells or at least might represent a contributing factor. The finding that PLCß1, although overexpressed in cord T-cells, is unable to rescue the low inositol phosphate production might be explained by the idea that the activation of PLCß isoforms in T-cells is dependent on a reciprocal regulation between tyrosine kinases and G-proteins (
1 in response to antigen stimulation (
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altman LG, Sheider BG, Papermaster DS (1984) Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy. J Histochem Cytochem 32:1217-1223[Abstract]
Cocco L, Gilmour RS, Ognibene A, Manzoli FA, Irvine RF (1987) Synthesis of poliphosphoinositides in nuclei from Friend cells. Biochem J 248:765-770[Medline]
Dent CL, Latchmann DS (1992) In Latchmann DS, ed. Transcription Factors: A Practical Approach. Oxford, UK, FIRL Press
Ericsson PO, Orchansky PL, Carlow DA, Teh HS (1996) Differential activation of phospholipase C1 and mitogen-activates protein kinase in naive and antigen-primed CD4 T cell by the peptide/MHC ligand. J Immunol 156:2045-2053[Abstract]
GarciaBustoss JF, Marini F, Stevenson I, Frey C, Hall MN (1994) PIK1, an essential phosphatidylinositol 4 kinase associated with the yeast nucleus. EMBO J 13:2353-2361
Gauen LKT, Zhu YX, Letourneur F, Hu Q, Bolen JB, Matis LA, Klausner RD, Shaw AS (1994) Interactions of p59fyn and ZAP70 with T-cell receptor activation motifs: defining the nature of a signalling motif. Mol Cell Biol 14:3729-3741[Abstract]
Han P, Hodge G, Story C, Xu X (1995) Phenotypic analysis of functional T lymphocyte subtypes and natural killer cells in human cord blood: relevance to umbilical cord blood transplantation. Br J Haematol 89:733-740[Medline]
Harris DT, Schumacher MJ, Locascio J, Besencon FJ, Olson GB, De Luca D, Shenker L, Bard J, Boyse EA (1992) Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes. Proc Natl Acad Sci USA 89:10006-10010[Abstract]
Jayaraman T, Ondriasova E, Ondrias K, Harnick DJ, Marks AR (1995) The inositol 1,4,5-trisphosphate receptor is essential for T cell receptor signalling. Proc Natl Acad Sci USA 92:6007-6011
Marmiroli S, Ognibene A, Bavelloni A, Cinti C, Cocco L, Maraldi NM (1994) Interleukin 1 stimulates nuclear phospholipase C in human osteosarcoma SaSOS2 cells. J Biol Chem 269:13-16
Martelli AM, Gilmour RS, Bertagnolo V, Neri L, Manzoli L, Cocco L (1992) Nuclear localization and signaling activity of phosphoinositidase C ß in Swiss 3T3 cells. Nature 358:242-244[Medline]
Miscia S, Di Baldassarre A, Rana R, Cataldi A (1997) Phospholipase C gamma 1 overexpression and activation induced by interferon beta in human T lymphocytes: an ISGF3-independent response. Cytokine 9:660-665[Medline]
Payrastre B, Nievers M, Boonstra J, Breton M, Vrkleij AJ, Vanbergenen Henegouwen PMP (1992) A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix. J Biol Chem 267:5078-5084
Samelson LE, Klausner RD (1992) Tyrosine kinases and tyrosine-based activation motifs. J Biol Chem 267:24913-24916
Sieh M, Batzer A, Schlessinger J, Weiss A (1994) GRB2 and phospholipase C gamma1 associate with a 36 to 38 kilodalton phosphotyrosine protein after T cell receptor stimulation. Mol Cell Biol 14:4435-4442[Abstract]
Stanners J, Kabouridis PS, McGuire KL, Tsoukas CD (1995) Interaction between G proteins and tyrosine kinases upon T cell receptor CD3-mediated signalling. J Biol Chem 270:30635-30642
Taylor S, Bryson YJ (1985) Impaired production of -interferon by newborn cells in vitro is due to a functionally immature macrophage. J Immunol 134:1493
Tucci A, Mouzaki A, James H, Bonnefoy JY, Zubler RH (1991) Are cord blood B cells functionally mature? Clin Exp Immunol 84:389-394[Medline]
Weber JR, Bell GM, Han MY, Pawson T, Imboden JB (1992) Association of the tyrosine kinase LCK with phospholipase C gamma 1 after stimulation of the T cell antigen receptor. J Exp Med 176:373-379[Abstract]
Wilson CB (1991) The ontogeny of T lymphocyte maturation and function. J Pediatr 118:S4[Medline]