The expanding roles of ITAM adapters FcRgamma and DAP12 in myeloid cells

Abstract

The adapter proteins DAP12 and FcRgamma associate with a wide spectrum of receptors in a variety of innate immune cells to mediate intracellular signaling pathways when their cognate receptor is engaged. These adapter proteins are coupled to their receptors through charged residues within the transmembrane regions of the adapter and receptor. DAP12 and FcRgamma contain specific protein domains (referred to as immunoreceptor tyrosine-based activation motifs) that serve as the substrates and docking sites for kinases, allowing amplification of intracellular signaling reactions. Recent research has broadened the repertoire of receptors that utilize these adapters for signaling to include not only novel immunoglobulin superfamily members but also cytokine receptors, integrins, and other adhesion molecules. There is abundant evidence that these multifunctional signaling adapters also mediate inhibitory activity, downmodulating signaling from Toll-like receptors and other heterologous receptors. In this review, we discuss the newly described receptors that utilize DAP12 and/or FcRgamma adapters to modulate innate immune responses.

Figure 1. Activating and inhibitory pathways initiated by DAP12 and FcRγ associated receptors

The left hand panel indicates the downstream signaling pathways that lead to activation of cellular responses when DAP12 and/or FcRγ associated receptors are engaged with high affinity/high valency ligands. The receptors associated specifically with either DAP12 or FcRγ that are discussed in the text are listed, with the immunoreceptors indicated in black font and the non-immunoreceptors in gold font. Dually associated receptors are indicated in blue font. For a more exhaustive list of adapter-associated receptors see Lanier (4) and Nimmerjahn (5). Based on the model of Monteiro (80) the potential mechanism of inhibitory function of DAP12 and FcRγ associated receptors is shown on the right side. These functions would become manifest under low affinity/avidity (ie monovalent) ligand interaction with the receptor(s), which would induce weak phosphorylation of the DAP12 and/or FcRγ ITAMs, leading to primarily SHP-1 phosphatase recruitment. SHP-1 blocks downstream pathways from other DAP12/FcRγ associated receptors or from completely heterologous receptors, such as the TLRs. The inhibitory function is indicated by blue and red lines terminating in rosettes. Whether the same model of low affinity/avidity interactions, leading to SHP-1 recruitment also mediate the trans-inhibitory function of the DAP12/FcRγ associated receptors on TLRs (and other receptors) remains unclear, and hence is indicated by a question mark.

Figure 2. DAP12/FcRγ or Syk-deficient dendritic cells show reduced proliferative responses and increased apoptosis to GM-CSF stimulation

(A) 1 × 10⁶ WT or mutant bone marrow cells were cultured in the presence of 10 ng/ml GM-CSF (Peprotech) for the indicated days and number of CD11c+ cells was determined by flow cytometry and hematocytometer counts of viable cells. Results are the mean +/− SD of 3 wells. (B) WT or mutant bone marrow cells were cultured as in A. At the indicated days, the number of CD11c+ apoptotic cells was determined by annexin V staining and flow cytometry.

Figure 3. DAP12-deficient macrophages have increased phagocytic capacity and are hyperresponsive to IFNγ stimulation

(A) Bone marrow macrophages were grown as described (90). WT and DAP12-deficient bone marrow derived macrophages (2 × 10⁵ cells) were incubated with AlexaFluor488-labeled, IgG-opsonized SRBCs for 20 minutes. Unbound SRBCs were removed by washing in cold PBS, then bound but extracellular SRBCs were lysed by incubation of the macrophages in water for 1 min, followed by PBS wash and fixation in 4% paraformaldehyde in PBS. The percentage of macrophages that had phagocytosed the labeled SRBCs was measured by flow cytometry. (B) WT and DAP12-deficient bone marrow derived macrophages (2 × 10⁵ cells) were incubated with FITC-labeled zymosan particles for 20 minutes. Unbound zymosan was removed by washing in PBS, then bound but extracellular zymosan was removed by incubation with proteinase K (2.5 units/ml) for 20 minutes at 25⁰C. Phagocytosis of zymosan was measured by flow cytometry. (C) Day 6 WT and DAP12-deficient bone marrow derived macrophages (2 × 10⁵ cells) were incubated with the indicated concentrations of rmIFNγ for 48 hours at 37⁰C, blocked with 2.4G2 mAb and then stained with FITC-labeled anti-MHCII mAb, followed by flow cytometry.

Figure 4. DAP12-deficient macrophages display increased activation of Akt following TLR9 stimulation

WT and DAP12-deficient bone marrow-derived macrophages were incubated with 25 nM CpG oligonucleotides (CpG ODN 1826, Invivogen) for the indicated time (min). Cells were then lysed and 5 μg of cytoplasmic extract for each time point was subjected to western blotting with polyclonal anti-phospho Akt antibody. The blot was then stripped and reprobed with an antibody to total Akt to confirm equal loading.