Preparation of intact, highly purified phagosomes from Dictyostelium.

Phagocytosis plays a fundamental role in the immune system for the defense against invading microorganisms and the clearing of apoptotic and cancerous cells. The common amoeba Dictyostelium discoideum is a recognized model for professional immune phagocytes and is now commonly used to study host-pathogen interactions. Dictyostelium is genetically and biochemically tractable and is a most versatile experimental system. The classical protocol for purifying phagosomes formed by ingestion of latex beads particles has been adapted to Dictyostelium. It was improved in yield, purity, and synchronicity, allowing isolation of milligram amounts of phagosomal proteins and lipids. This method has been used successfully to highlight membrane trafficking and phagosome maturation. Here, we present a step-by-step protocol including detailed notes necessary for ensuring access to a large number of highly synchronized phagosomes of high purity and integrity.

Cognate MHC-TCR interaction leads to apoptosis of antigen-presenting cells.

Antigen presentation to T lymphocytes has been characterized extensively in terms of T lymphocyte activation and eventual cell death. In contrast, little is known about the consequences of antigen presentation for the antigen-presenting cell (APC). We have determined the outcome of major histocompatibility complex class II-restricted peptide presentation to a specific T cell. We demonstrate that specific T lymphocyte interaction with peptide-presenting APCs led to apoptosis in the APC population. In contrast, T lymphocyte interaction with nonpeptide-loaded APCs or APCs loaded with monosubstituted peptide failed to induce T lymphocyte secretion of interleukin-2 and APC apoptosis. Phosphatidylserine externalization and mitochondrial depolarization were used to evaluate APC apoptosis. Fas/Fas ligand interactions were not required, but cytoskeletal integrity and caspase activation were essential for APC apoptosis. Antigen presentation leading to T lymphocyte activation is therefore coordinated with apoptosis in the APC population and could provide a mechanism of immune response regulation by eliminating APCs, which have fulfilled their role as specific ligands for T lymphocyte activation. This pathway may have particular importance for APCs, which are not sensitive to death receptor-induced apoptosis.

HLA class II signals sensitize B lymphocytes to apoptosis via Fas/CD95 by increasing FADD recruitment to activated Fas and activation of caspases.

Human leukocyte antigen (HLA) class II heterodimers have a well defined role in peptide presentation to helper T lymphocytes. Moreover, engagement of HLA class II molecules leads to signal transduction in the antigen presenting cell. Signaling via HLA-DR increases CD95 mediated hypoploidy in B-cell blasts. Given the importance of CD95 for the homeostasis of lymphocyte populations, we examined the impact of the HLA-DR signal on the most proximal events in the CD95 apoptotic pathway. CD95 activation recruits the adapter molecule Fas-associated death domain protein (FADD) and thereby provides a scaffold for procaspase-8 activation. The HLA-DR signal increased both recruitment of FADD and activation of caspases-3 and -8 via CD95 activation. Sensitization was tightly controlled because neither FADD recruitment to CD95 nor caspase activation was induced via HLA-DR alone. In contrast, the HLA-DR signal and the CD95 signal both led to decreased mitochondrial membrane potential. Taken together, these data indicate that ligand interaction with HLA class II molecules preactivates the antigen presenting cell for death in the event of a subsequent interaction with the CD95 ligand. This would ensure termination of a specific immune response, particularly in cells with limited sensitivity to CD95 mediated apoptosis alone.

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake.

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Galpha4 and Gbeta, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation.

Stimulation of Toll-like receptors (TLRs) initiates potent innate immune responses through Toll-interleukin 1 receptor (TIR) domain-containing adaptors such as MyD88 and Trif. Analysis of Trif-deficient mice has shown that TLR3-dependent activation of the transcription factor NF-kappa B by the TLR3 ligand double-stranded RNA is Trif dependent. Here we investigated the 'downstream' signaling events that regulate TLR3-dependent Trif-induced NF-kappa B activation. Trif recruited the kinases receptor interacting protein (RIP)-1 and RIP3 through its RIP homotypic interaction motif. In the absence of RIP1, TLR3-mediated signals activating NF-kappa B, but not the kinase JNK or interferon-beta, were abolished, suggesting that RIP1 mediates Trif-induced NF-kappa B activation. In contrast, the presence of RIP3 negatively regulated the Trif-RIP1-induced NF-kappa B pathway. Therefore, in contrast to other TLRs, which use interleukin 1 receptor-associated kinase (IRAK) proteins to activate NF-kappa B, TLR 3-induced NF-kappa B activation is dependent on RIP kinases.

MHC class II-mediated apoptosis of mature dendritic cells proceeds by activation of the protein kinase C-delta isoenzyme.

The mature dendritic cell (DC) is considered to be the most potent antigen-presenting cell. Regulation of the DC, particularly its survival, is therefore critical. Mature DC are markedly more sensitive to HLA-DR-mediated apoptosis than immature DC. To further characterize this key survival difference, we compared the intracellular signals initiated via HLA-DR in mature versus immature DC. Apoptosis was unchanged by inhibition of tyrosine kinases or phosphatases. HLA-DR-mediated re-localization of protein kinase C (PKC)-delta to the nucleus was detected in mature DC by confocal microscopy and by immunoblotting. Activation of PKC-delta in mature DC was revealed by the detection of the PKC-delta catalytic fragment in the nuclear fraction isolated from mature DC which had been stimulated via HLA-DR. The broad-spectrum PKC inhibitor, Calphostin C, as well as the PKC-delta-selective inhibitor, Rottlerin, inhibited HLA-DR-mediated apoptosis of mature cells. Taken together, these data reveal a role for the PKC-delta isoenzyme in regulating HLA class II-mediated apoptosis of mature DC. Thus, the lifespan of the mature DC could be controlled by signals generated in the course of antigen presentation, and thereby prevent DC persistence and prolonged stimulation of T and B lymphocytes.