Abstract. Human, but not murine, renal peritubular and glomerular capillaries constitutively express class II major histocompatibility (MHC) proteins at high levels in normal human kidney. Expression of class II proteins on renal microvascular endothelial cells (RMEC) makes it available to circulating lymphocytes and imparts a surveillance capacity to RMEC for controlling inflammatory responses. In this report, the coexpression of HLA-DR and the endothelial marker CD31 are used to identify RMEC as a distinct population of cells within a standard renal biopsy using flow cytometry. A three-laser, multicolor flow cytometry analysis using Alexa dyes, developed for characterizing the expression of cell surface antigens, identifies RMEC as a population separate from HLA-DRexpressing leukocytes. HLA-DR RMEC co-express HLA-DP and HLA-DQ. RMEC also express the T cell costimulatory factor CD58 but not CD80, CD86, or CD40. On the basis of high HLA-DR expression, RMEC are isolated for culture using fluorescence-activated cell sorting and magnetic beads. Cultured RMEC require normal basal physiologic concentrations of gamma interferon (␥IFN) to maintain HLA protein expression. This expression is regulated by CIITA, the MHC class II-specific transcription factor. Four tissue-specific promoters have been described for CIITA. In freshly isolated RMEC, RT-PCR and hybridization using specific oligonucleotide probes to CIITA promoter sequences identify only the statinsensitive ␥IFN-induced promoter IV of CIITA. Therefore, the constitutive expression of HLA-DR on RMEC in normal human kidney is located in a position for immune surveillance, depends on basal physiologic concentrations of ␥IFN, and may be amenable to regulation with statins.MHC proteins are of two classes distinguished on the basis of structure and function. Class I molecules, composed of a polymorphic subunit complexed with ␤-2-microglobulin, are found on all nucleated cells and present antigenic peptides to CD8ϩ T lymphocytes; class II molecules, composed of polymorphic ␣ and ␤ chains, are constitutively expressed on a limited number of cell types (dendritic cells, macrophages, B lymphocytes) and present antigenic peptides to CD4 ϩ T lymphocytes. We recently described an unusual expression of MHC class II proteins in normal human kidneys that is not found in murine kidneys (1). The human MHC class II protein HLA-DR is abundantly expressed on peritubular and glomerular capillary endothelial cells but not on endothelial cells of larger blood vessels of normal kidney. Antibodies to HLA-DR and CD31, a protein highly expressed on endothelial cells, co-localize on peritubular and glomerular cells within sections of kidney tissue, indicating capillary endothelial cell location of HLA-DR. HLA-DR has also been identified on rare scattered circulating leukocytes found within the kidney, but over 98% of the DR identified by immunofluorescence microscopy in kidney cortex is located on capillary endothelial cells (1). We refer to these cells co-expressing HLA-DR and CD31 as rena...

Background: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented. Methods: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer. Four single color fluorescent control samples of cells were run to quantify spectral overlap. An additional sample, labeled with all colors was run and compensated in six spectral channels. Results: Analysis of empirical data for sensitivity and dynamic range matched theoretical predictions. The ImageStream system demonstrated fluorescence sensitivity comparable to a PMT-based flow cytometer. A methodology for addressing spectral overlap, individual pixel ano-

Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes or the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre‐fixation viability of fixed cells either before or after fixation with amine‐reactive dyes suitable for a range of excitation wavelengths. Membrane‐impermeable dead cell and live cell dyes as well as dye‐exclusion procedures for microscopy are also included. Curr. Protoc. Cytom. 64:9.2.1‐9.2.26. © 2013 by John Wiley & Sons, Inc.

SummaryTrypanosoma cruzi is an obligate intracellular protozoan parasite. The mammalian stage of the parasite life cycle describes amastigotes as an intracellular form that replicates, and trypomastigotes as an extracellular form that disseminates and invades cells. Recent studies, however, have demonstrated that amastigotes circulate in the blood of infected mammals and can invade mammalian cells. In this report, a T. cruzi surface glycoprotein gene, SA85-1.1, was expressed as an immunoglobulin chimera, and this recombinant globulin was used to screen normal mouse tissues for adhesive interactions. This approach identified a subset ofmacrophages in the skin and peripheral lymph node that bind the T. cruzi surface glycoproteins through the mannose receptor. To further examine the T. cruzi mannose receptor carbohydrate ligands, the interaction between T. cruzi and the mannose-binding protein, a mammalian lectin with similar carbohydrate binding specificities as the mannose receptor, was examined. These studies demonstrated that the mannose-binding protein recognized amastigotes, but not trypomastigotes or epimastigotes, and suggested that amastigotes would also be recognized by the rnannose receptor. Therefore, amastigote adhesion to macrophages was investigated, and these experiments demonstrated that the mannose receptor contributes to amastigote adhesion. The data identify the first mammalian lectins that bind to T. cruzi, and are involved in T. cruzi invasion of mammalian cells. The data suggest that amastigotes and trypomastigotes may have developed different mechanisms to adhere to and invade host cells. In addition, it has been established that IFN-',/-activated macrophages express low levels of the mannose receptor and are trypanocidal; this suggests that the interaction between amastigotes and the mannose receptor enables amastigotes to increase their adherence with a population ofmacrophages that are nontrypanocidal and permissive for their intracellular replication.

All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

Recently, it has been demonstrated that stimulated T cells bearing defects in caspase-8 fail to promote nuclear shuttling of NF-κB complexes. Such cells display strikingly similar proliferative and survival defects as T cells lacking Fas-associated death domain protein (FADD) function. We characterized NF-κB signaling in T cells bearing a dominant-negative FADD transgene (FADDdd). Whereas FADDdd T cells displayed proliferative defects following activation, these were not a consequence of aberrant NF-κB signaling, as measured by IKK/IκB phosphorylation and IκB degradation. There were no appreciable defects in nuclear translocation of p65/Rel using ImageStream, a flow-based imaging cytometer. Pretreatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a potent caspase inhibitor, also failed to impede canonical NF-κB signaling. Secretion of IL-2 and up-regulation of various activation markers occurred normally. Thus, FADD does not play an essential role in NF-κB activation, suggesting an alternative route by which this adaptor promotes the clonal expansion of T cells.

Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes of the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre‐fixation viability of fixed cells either before or after fixation. A dye‐exclusion procedure for microscopy is also included.

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