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Gap junction channels constructed of connexins (Cxs) are expressed by peripheral and secondary lymphoid organ-derived lymphocytes. These channels in the plasma membrane play key roles in a range of lymphocyte functions exemplified by the synthesis and secretion of Igs and cytokines and during transmigration across the endothelium. Most recently, their involvement in antigen cross-presentation has also been established. We report here for the first time the expression of mRNA and protein encoding Cx43 in mouse-derived CD4+ Th0, Th1, and Th2 lymphocyte subpopulations and demonstrate the establishment gap junction channel formation with primary macrophages in vitro. We show that this mode of direct communication is particularly favored in Th1-macrophage interactions and that LPS inhibits lymphocyte-macrophage cross-talk independently of the subset of lymphocyte involved. Our work suggests that gap junction-mediated communication can be modulated in the absence of specific antigenic stimulation. Therefore, a further mechanism featuring gap junction-mediated communication may be implicated in immune regulation.
Intercellular communication underpins cellular activation and various functions in mammals. In the hematopoietic and immune systems, direct cell-to-cell interactions influence cell phenotypic and functional characteristics such as those involved in blood formation or antigen-specific immune responses. These processes involve subsets of interacting cells, the surrounding signaling environment, and the functional outcomes of receptor-ligand interactions. One of the key channels underpinning intercellular communication are gap junctions, which are the focus of research by various groups who have shown their participation in leukocyte biology and the generation of immune responses [1-4].
Gap junctions are plasma membrane channels, which directly link the cytoplasms of attached cells. This communication pathway consists of paired hexameric connexin hemichannels (CxHc) or connexons assembled from individual subunits, called connexins (Cxs), arranged around a central pore. The gap junction channel allows bidirectional exchange of ions and molecules of 1–1.5 kDa , such as Ca2+, cAMP, D-myo-inositol-1,4,5-trisphosphate, and NAD+, as well as ATP, glucose, amino acids, and peptides [3, 6]. CxHc can be formed as one (homomeric) or more (heteromeric) Cx protein subunits, thus establishing, after docking, homotypic and/or heterotypic gap junction intercellular pathways endowed with varying molecular selectivities. The functionality of these channels is determined by intracellular and extracellular Ca2+ levels and electrical membrane potentials among others [5, 6].
The expression of Cxs by T, B, and NK cells derived from peripheral blood and secondary lymphoid organs has been reported [7-12]. Circulating lymphocytes express mainly Cx43, whereas expression of Cx40 occurs mainly in lymphocytes derived from secondary lymphoid organs . Interruption of direct intercellular communication between lymphocytes leads to important functional consequences such as the inhibition of the synthesis and secretion of Igs and cytokines such as IFN-γ, IL-2, and IL-10 . Cx43 has also been suggested to play a key role in leukocyte-endothelium communication during cell transmigration .
Despite increasing evidence for the role of Cx proteins and gap junction channels in inflammatory and immunological reactions, their expression and specific functional roles remain to be studied in lymphocyte subsets with distinct functional properties. Here, we describe for the first time the differential expression of mRNA and protein encoding Cx43 in mousederived CD4+ Th lymphocyte subpopulations in vitro. We show that all of these cells can communicate with macrophages via gap junctions and that such cross-talk is particularly favored in Th1-macrophage interactions.
A well-established methodology was used to obtain differentiated CD4+ Th1 and Th2 lymphocyte subpopulations in the absence of antigenic stimulation using Th0 (naïve) lymphocytes isolated from the spleen of specific pathogen-free (SPF) CD-1 male mice . Naïve CD4+ T cells were obtained by negative selection from the spleens of 8- to 10-week-old SPF male CD-1 mice (Harlam, UK). B cells, NK cells, monocytes, and CD8+ T cells were removed by incubating splenocytes with rat anti-mouse CD19, CD11c, and CD8 mAb (Serotec, UK) and anti-CD16/32 (PharMingen, San Diego, CA, USA) before incubation with goat anti-rat IgG-coated beads (BioMag, Qiagen, UK), according to the manufacturer’s instructions. T cell differentiation was induced by culturing 1-5 × 106 isolated CD4+ cells in rat anti-mouse CD3 (Clone 17A2, 2 μg/ml, eBiosciences, San Diego, CA, USA)-coated, 96-well plates. In addition, Th1 cell culture media included 5 ng/ml IL-12 and 10 μg/ml anti-IL-4, whereas Th2 cultures were grown in media containing 5 ng/ml IL-4, 20 ng/ml anti-IL-12, and 20 ng/ml anti-IFN (Clone 37895.11). Cytokines were obtained from R&D Systems (UK) and antibodies from Serotec. After 3 days in culture, cells were harvested for analysis or coculture experiments. Culture supernatants were analyzed for cytokine secretion using ELISA. Production of IFN-γ and IL-5 by T cells was monitored using mouse ELISA kits from R&D Systems. Th0 cells and differentiated cells were used to assess the expression of mRNA encoding Cx43 by semiquantitative PCR. RNA isolation and first-strand cDNA synthesis were carried out as described previously . PCR analysis was performed in a final volume of 20 μl using 10 μl PCR Master Mix (Promega, Madison, WI, USA), 2 μl cDNA, and 20 pmol sense and antisense oligonucleotide primers for Cx43 (sense primer: 5′-TTC TAT GTT TTC TTC AAG GGC GTT AA-3′; antisense primer: 3′-TTG CTT GCT TGT TGT AAT TGC G-5′). The primers used for GAPDH were sense primer: 5′-ACC CAG AAG ACT GTG GAT GG-3′; antisense primer: 3′-CAC ATT GGG GGT AGG AAC AC-5′. The conditions for amplification were denaturation, one cycle at 94°C for 1 min; 30 amplification cycles each of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min; and one final extension cycle of 72°C for 4 min. The results showed a significant up-regulation of Cx43 by Th1 cells (5.51±0.4; P<0.009) with respect to Th0 lymphocytes (3.08±0.3) and to Th2 lymphocytes (0.2±0.07). Detection of Cx43 mRNA in a freshly processed mouse heart ventricular muscle was used as an internal positive control. Primary mouse hepatocytes (Tebu-Bio 158DPK-HCWP-M) were used as negative controls (Fig. 1). These results correlated with those obtained from Western blot analysis. Th0 or differentiated cells were resuspended in PBS (pH 7.4) and lysed by vortexing after resuspension in a lysis buffer, pH 7.4, containing 100 mM Tris-HCl, 20 mM EDTA, protease inhibitors (protease inhibitors cocktail, Sigma, UK), and 1% SDS. Lysates were then mixed with SDS-loading buffer, and equal amounts of total protein were loaded onto 12.5% (w/v) SDS polyacrylamide gels. After electrophoresis, proteins were transferred onto nitrocellulose membranes, blocked, and then incubated overnight with an anti-Cx43 mAb (Chemicon International, El Segundo, CA, USA). A mouse GAPDH mAb (mAbcam 9484) was used in a separate blot as a loading control. After incubation with HRP-conjugated anti-mouse IgG and repeated washings, antigen-antibody recognition was detected by ECL (Amersham, UK). Relative protein amount was measured by densitometric analysis and expressed as arbitrary units. Th1 lymphocytes also showed significantly higher expression of Cx43 (47.74±2.03; P<0.006). Cx43 expression by Th2 lymphocytes (26.5±1.3) was not significantly different than that shown by Th0 cells (Fig. 2).
A functional in vitro assay was used to evaluate whether Th0, Th1, and Th2 lymphocyte cell subsets were capable of establishing gap junction intercellular communication with macrophages and to test the hypothesis of whether the observed mRNA and protein expression levels also correlated with the degree of intercellular communication. A methodology described previously [7, 9] was based on the assessment of transfer of a gap junction permeant green fluorescent dye, calcein AM, between cells. First, we used DiIC18, a red fluorescent dye, which does not move across gap junction channels, to label each lymphocyte subset, and then cocultures were set up to study dye transfer from macrophages into red-labeled (DiIC18-loaded) lymphocyte subsets. Macrophages were obtained by peritoneal lavage of mice with RPMI-1640 medium (Gibco-BRL, UK) containing 1 × 105 U penicillin and 1 × 105 U streptomycin. Cells were pooled and after removing contaminating RBC (erythrocyte lysis kit, Promega, UK), they were adjusted to 1–2 × 106 cells/ml. Macrophage purity was assessed by flow cytometry analysis using rat anti-mouse CD4, CD19, CD11c, and CD8 mAb (all from Serotec). Cell morphology and purity were also assessed by staining slide-mounted peritoneal cell samples with rat anti-mouse F4/80 (Serotec; data not shown). This method produced samples of more than 90% purity, and the viability of cells before dye loading was 90-94%. Macrophages were then used for coculture experiments and dye transfer analysis. Briefly, each lymphocyte subset was incubated for 30 min at 37°C with 10 μM DiIC18 and macrophages with 2.5 μM calcein AM in RPMI 1640. The cells were washed extensively with a solution of PBS/BSA 1% and cocultured at a ratio of 1:10 (macrophage:lymphocyte). After 30 min in coculture, lymphocytes were harvested, washed in PBS/BSA 1%, and dye transfer-assessed by flow cytometry as described previously [7, 9]. Data from 2 × 104 events were collected, and results were expressed as the percentage of double-labeled cells detected within the specified region determined using parameters acquired from control reactions of single-labeled cells. The specificity of dye transfer across gap junction channel samples was carried out using cocultures set up in parallel but in the presence of 150 μM 18-α-glycyrrhetinic acid (AGA). Communication was assessed by flow cytometry 30 min after the cells were cocultured. Figure 3 shows that dye transfer occurred in all groups and that it was higher from macrophages to Th1 lymphocytes when compared with transfer to Th0 or Th2 cells. The gap junction channel inhibitor, AGA, was used to assess the specificity of the communication through gap junction channels. AGA showed a small blocking effect on calcein dye transfer from macrophages to Th0 cells intercellular dye transfer (0.89% reduction of calcein transfer), but the blocking effect was greater when added to macrophage-Th1-cocultured cells (8.93% reduction of calcein transfer). The effects of AGA were also greater with macrophage-Th2 cell cocultures (5.61% reduction of calcein transfer). These results are similar to data reported previously about the levels of gap junction intercellular communication blockage by AGA in cocultures set up between T and B lymphocytes in the absence of specific antigenic stimulation .
Previous work has also shown that the level of gap junction intercellular communication was modulated by polyclonal stimulators such as bacterial LPS . Similar cocultures to those described above were set up, but they were supplemented with LPS to investigate whether this effect could also affect the level of gap junction-mediated intercellular communication between CD4+ T lymphocyte subsets and macrophages. In contrast to previous results, LPS showed an inhibitory/blocking effect on calcein dye transfer between macrophages and lymphocytes (Fig. 4), which was dose-dependent (data not shown). However, LPS has been reported previously to have similar inhibitory/blocking effects in the context of inflammatory reactions in vitro. LPS may cause dose-dependent inhibition of processes such as macrophage 5-lipoxygenase metabolism , inhibition of the expression of macrophage scavenger receptors [15, 16], interference with macrophage viral infection , and inhibition of hormone secretion [18-20]. LPS can also differentially regulate CxHc responses in pathological, stimulatory conditions . Another mechanism proposed to explain the effects of LPS involves activation of monocyte/macrophage NO synthase and/or the induction of the release of important anti-inflammatory cytokines by macrophages such as IL-10 .
Altogether, the present results illustrate the importance of gap junction intercellular channels in immune-mediated processes, and they reinforce the hypothesis that these channels may function in the immunological synapse. The present studies also show that gap junction-mediated communication can be modulated in the absence of antigenic stimulation. In addition, they suggest an alternative route occurring for the maintenance of classical mechanisms of immune surveillance, as shown by studies showing that gap junction channels constructed of Cx43 participate in antigen cross-presentation, underpinning immune responses such as those involved in infection or tumorigenesis [2, 3]. Indeed, gap junctions have been suggested to provide direct “information-sharing” conduits for the transfer of viral- and tumor-derived peptides between professional APC and naïve T lymphocytes, allowing further stimulation and modulation of immune responses . The present results also raise questions about the role of gap junction channels in other processes such as the maintenance of immunological tolerance or their participation in the polarization of immune responses to antigens, which do not follow classical routes of processing and presentation.
This study was supported by a grant from the British Heart Foundation (FS/02/077/14753) to E. O-O. and A. C. N.