Regulation of hemichannels and gap junction channels by cytokines in antigen-presenting cells

Abstract

Autocrine and paracrine signals coordinate responses of several cell types of the immune system that provide efficient protection against different challenges. Antigen-presenting cells (APCs) coordinate activation of this system via homocellular and heterocellular interactions. Cytokines constitute chemical intercellular signals among immune cells and might promote pro- or anti-inflammatory effects. During the last two decades, two membrane pathways for intercellular communication have been demonstrated in cells of the immune system. They are called hemichannels (HCs) and gap junction channels (GJCs) and provide new insights into the mechanisms of the orchestrated response of immune cells. GJCs and HCs are permeable to ions and small molecules, including signaling molecules. The direct intercellular transfer between contacting cells can be mediated by GJCs, whereas the release to or uptake from the extracellular milieu can be mediated by HCs. GJCs and HCs can be constituted by two protein families: connexins (Cxs) or pannexins (Panxs), which are present in almost all APCs, being Cx43 and Panx1 the most ubiquitous members of each protein family. In this review, we focus on the effects of different cytokines on the intercellular communication mediated by HCs and GJCs in APCs and their impact on purinergic signaling.

Figures

Figure 1

Connexin 43 and pannexin1 at gene and protein levels. Left: a diagram depicting the genomic regions, mRNA, and membrane topology of human connexin 43 (Cx43, top left) and pannexin 1 (Panx1, bottom left). Genomic loci are represented by black boxes that stand for the corresponding exons. mRNA diagrams representing the exons as coding protein regions (red boxes) and 3′- and 5′-non-coding areas (purple boxes) are shown. The intron lengths are indicated in the schemes of genomic loci, and exon sizes are indicated in the mRNA diagrams. In the membrane topology the white squares indicate extracellular cysteine residues of each protein. Six protein subunits constitute a hemichannel (HC), which has different pore sizes. Right: two adjoining cells forming a gap junction channel (GJC) at the cell interface. Each cell presents HCs formed by Cx43 or Panx1. Arrows denote the bidirectional communication with the intracellular milieu (ICM) for GJCs and the extracellular milieu (ECM) for HCs; some immunorelevant molecules are shown. Dotted line for Ca2+ permeating Panx1 HCs indicates that this phenomenon is not fully demonstrated.

Figure 2

Connexin and pannexin expression in antigen-presenting cells. Summary scheme shows the expression of gap junction channels (GJCs) and hemichannels (HCs) formed by connexins (Cxs) and pannexins (Panxs) in different antigen-presenting cells (APCs). Question marks next to a protein (Cx or Panx) or channel type (GJC or HC) indicate that the expression or function remains unknown or is not fully shown.

Figure 3

Dendritic and B cells of mouse spleen present pannexin1. Immunofluorescence of adult mice spleen cryosections (8 μm thick) fixed in ethanol (70% v/v) at −20°C for 20 min, mounted in Fluoromount-G and observed in a confocal microscope (Olympus, FluoView FV1000). Pannexin1 (Panx1 in green: primary antibody: rabbit anti-Panx1 antibody and secondary antibody goat anti-rabbit IgG conjugated to FITC) immunoreactivity is shown. Cells were identified by their reactivity to specific antigens as follows: dendritic cells (DCs) by CD11c (blue, monoclonal mouse antibody conjugated to allophycocyanin) and B cells by B220 (red, monoclonal mouse antibody conjugated to phycoerythrin) in a follicle. Arrows denote follicular DCs (arrows). Merge is also shown. Scale bar: 100 μm.

Figure 4

IFN-γ or IL-6 increases dye uptake in dendritic cells. Bone-marrow-derived dendritic cells (BMDCs) from balb/c mice were differentiated with 40 ng/mL GM-CSF and IL-4 for 7 days. At day 7, BMDCs were treated for 6 h with IFN-γ (10 ng/mL), IL-6 (10 ng/mL), or both, and ethidium uptake was evaluated in time-lapse experiments (Olympus BX 51W1I). (a) Left: fluorescence images of ethidium after 9 min of uptake. Scale bar: 50 μm. Right: ImageJ surface plot analysis of fluorescence intensity of the region indicated in the field (dotted square). (b) Top: time-lapse ethidium uptake under control conditions (white circles) or after 6 h treatment with IL-6 (yellow triangles) or IFN-γ (blue diamonds). Each point corresponds to the mean of 30 cells. After 10 min of recording, La3+ (200 μM) was added to the bath solution to block connexin hemichannels. Bottom: graph showing the basal dye uptake rate and the effect of La3+ on BMDCs after treatment with IFN-γ (blue bars), IL-6 (yellow bars), or both (green bars). Each bar corresponds to the mean ± SE (% of control condition, dotted line) of 3 independent experiments.

Figure 5

Expression of pannexin1 (Panx1) in Kupffer cells. Immunofluorescence analysis of liver cryosections (8 μm thick) obtained from adult wild-type (WT) and Panx1−/− adult (C57/BL6) mice was performed to analyze reactivity of Panx1 (green, primary antibody: rabbit anti-Panx1 antibody and secondary antibody goat anti-rabbit IgG conjugated to FITC) in ED2 (red: goat polyclonal antibody and secondary antibody mouse anti-goat conjugated to Cy3) positive cells, which correspond to Kupffer cells. Top panels correspond to a liver section of a WT mouse and bottom panels correspond to a liver section of a Panx1−/− mouse. No specific Panx1 reactivity was detected in Panx1−/− liver, but ED2 positive cells were evident. DAPI stain was used to visualize nuclei (blue), and merge is also shown. Panx1−/− mice were kindly donated by Dr. Hanna Monyer (University of Heidelberg, Germany). Bar: 10 μm.

Figure 6

B cells present pannexin1 at the cell surface. Confocal images (Olympus, FluoView FV1000) of immunofluorescence analysis of freshly isolated B cells fixed in ethanol (70%). B cells were isolated from peripheral lymph nodes by positive selection from balb/c mice. Top left: B cells were identified with IgG (conjugated to FITC, green); the inset shows the bright field. Top right: pannexin1 (Panx1) immunoreactivity (red, primary antibody: rabbit anti-Panx1 antibody and secondary antibody goat anti-rabbit IgG conjugated to Cy3) is shown. Bar: 20 μm. Middle left: using ImageJ colocalization finder, it can be seen that Panx1 colocalizes with IgG (white) at the cell surface in some B cells (white arrows). B cells with low or no colocalization are indicated (green arrows). Middle right: zoom and merge of IgG and Panx1 labeling in a B cell denoted by a dotted square at middle left panel. The white line denotes the region used for the line scan. Bar: 10 μm. Bottom: ImageJ line scan analysis shows the fluorescence intensity of each channel through the white line in the middle of each cell. The peak coincidence (denoted by dotted squares) is an index of colocalization between the different fluorophores.

Figure 7

Scheme of different stages of classical immune response. The reported role for connexin- and pannexin-based channels is depicted in different immune cells functions as migration, antigen presentation, clonal expansion, and apoptosis.