Dynamics of rab5 activation in endocytosis and phagocytosis

Fluid-phase endocytosis is stimulated by H-ras-linked growth factor receptors and this stimulation requires activation of rab5. We utilized a GFP-rab5a:wt fusion protein to monitor GFP-rab5a:wt activation in living fibroblasts and in J774 macrophages. Control CHO cells that expressed GFP-rab5a:wt were cultured in serum-free conditions and showed GFP-rab5a:wt localized to endosomal vesicles with a mean diameter of 0.3 +/- 0.1 microm. Endosome fusion, membrane ruffling, and pinosome formation were rarely detected in these cells. Coexpression of H-ras:G12V, a constitutively active H-ras mutant that activates rab5a, in cells resulted in marked enlargement of labeled endosomes (mean diameter 0.7 +/- 0.2 microm) and large numbers of giant GFP-rab5a:wt-positive endosomes were present. Time-lapse recordings showed abundant fusion among giant labeled endosomes, and membrane ruffling and pinosome formation were commonly observed. Alterations in GFP-rab5a:wt endosome structure and activity in cells expressing H-ras:G12V were linked to rab5a activation because these changes were identical to those found in cells expressing GFP-rab5a:Q79L, a constitutively activated rab5a mutant. Furthermore, cells co-expressing H-ras:G12V and GFP-rab5a:S34N, an inactive rab5a mutant, exhibited no evidence of H-ras:G12V-induced endosome enlargement. To observe changes in endosome structure and activity that directly followed activation of GFP-rab5a:wt, we performed time-lapse recordings of cells cultured overnight in serum-free media after addition of EGF. EGF caused a rapid increase in endosome fusion and in membrane ruffling activity. Membrane ruffling was often associated with GFP-rab5a:wt-positive vesicle (pinosome) formation at the base of membrane ruffles. Endosome and pinosome fusion were common in EGF-stimulated cells. Phagocytosis is also regulated by GFP-rab5a:wt. J774 macrophages that expressed GFP-rab5a:wt showed transiently activation and recruitment of GFP-rab5a:wt to newly formed phagosomes that contained rhodamine-labeled Escherichia coli. These studies show that GFP-rab5a:wt activation results in dynamic alterations in the structure and activity of the early endosomal and early phagosomal elements.     

Mutation in the DC-SIGN cytoplasmic triacidic cluster motif markedly attenuates receptor activity for phagocytosis and endocytosis of mannose-containing ligands by human myeloid cells

The transmembrane C-type lectin, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), has three conserved cytoplasmic tail motifs: the tyrosine (Y)-based, dileucine (LL), and triacidic cluster (EEE), which are believed to regulate ligand binding, uptake, and trafficking. We mutated each of these motifs by alanine substitution and tested their roles in phagocytosis and receptor-mediated endocytosis of the highly mannosylated ligands, Mycobacterium tuberculosis mannose-capped lipoarabinomannan (ManLAM) and HIV-1 surface glycoprotein gp120, respectively, in transfected human myeloid K-562 cells. Compared with wild-type and other mutants, the EEE mutant of DC-SIGN showed a reduced cell-surface expression, near abolishment in the phagocytosis of ManLAM-coated beads (90.5+/-0.4%), and a marked reduction in the endocytosis of soluble gp120 (79.3+/-0.7%). Although, the Y mutant of DC-SIGN did not exhibit any effect on phagocytosis and intracellular trafficking to the phagolysosome, the LL mutant caused the majority of the receptor and/or ligands to remain bound to the cell surface, indicating a role for the LL motif as an internalization signal. The majority of the EEE mutant protein was found to be retained by the intracellular trans-Golgi network and not by the late endosomal/lysosomal compartment of transfected K-562 cells. Collectively, our data indicate a dual role for the EEE motif as a sorting signal in the secretory pathway and a lysosomal targeting signal in the endocytic pathway.     

Differential requirements for Fgf3 and Fgf8 during mouse forebrain development

Multiple Fgfs are expressed during formation and patterning of the telencephalon in vertebrates. Fgf8 has been shown to control the size of the telencephalon and the development of signaling centers in zebrafish and mouse. Next to Fgf8, Fgf3 also influences telencephalic gene expression in the zebrafish. Moreover, Fgf3 and Fgf8 have been shown to have combinatorial functions during forebrain development in this species. Here, we have examined telencephalic development in Fgf3 null mouse mutants and embryos that lack both Fgf3 and Fgf8 in their forebrain. In contrast to zebrafish, Fgf3 mutants show normal forebrain development and expression of telencephalic marker genes. Although double mutants for Fgf3 and Fgf8 show a further reduction of forebrain size no additional changes of telencephalic gene expression are observed compared with Fgf8 mutants. Therefore unlike in zebrafish, Fgf3 is not required for mouse forebrain development whereas Fgf8 has a central role during this process. Developmental Dynamics 237:3417–3423, 2008. © 2008 Wiley‐Liss, Inc.     

The effect of interleukin 2 on Fc-gamma receptor function of human monocytes requires specific intercellular interaction

We examined the effect of interleukin 2 (IL 2) on the capacity of human monocytes to secrete reactive oxygen species triggered via Fc-gamma receptor (Fc gamma R) function as measured by chemiluminescence (CL). IL 2 did not activate highly purified (hp) monocytes to respond to Fc gamma R-mediated phagocytic stimulation with an enhanced respiratory burst activity, unless low numbers of T lymphocytes had been cocultured with hp monocytes. The degree of monocyte activation in peripheral blood mononuclear cells (PBMC) by IL2 was dependent on T lymphocytes to monocytes ratios as well as on IL 2 concentrations. Optimal Fc gamma R function was detected with IL 2 concentrations of 60 to 600 U/ml and one to two T lymphocytes per monocyte, whereas higher cellular ratios were suppressive. Supernatants from IL 2-treated PBMC contained interferon-gamma (IFN-gamma) and monocyte activating factor (MAF) activity (= enhanced monocytic Fc gamma R mediated CL). The secretion of both cytokine activities was strongly enhanced by a cooperative function of monocytes. The correlation of IL 2-induced secretion of IFN-gamma and MAF activity was striking; however, monoclonal anti-IFN-gamma antibodies failed to abrogate IL 2-stimulated and lymphocyte-dependent monocyte activation. Although IL 2 had no direct monocyte activating effect, pretreatment of hp monocytes with IL 2 led to a monocyte priming: subsequent coculture with autologous control T lymphocytes enhanced the monocyte Fc gamma R-mediated CL response. The priming of monocytes by IL 2 was dependent on the interaction of IL 2 with the monocytic IL 2 receptor, as shown by inhibition experiments with anti-IL 2 receptor monoclonal antibodies. Thus, we observed an IL 2-driven monocyte/T lymphocyte interaction leading to an increased Fc gamma R-mediated monocytic respiratory burst activity and to the secretion of a soluble MAF activity, but there were no detectable amounts of IFN-gamma.     

Differential stimulatory requirements and regulation of naive and primed human lymphocytes

In these investigations, human lymphocytes primed in vitro in MLR have been employed as a model for human memory cells and have been compared to naive lymphocytes from the same donor. Both the stimulatory requirements and the regulation of these cells were found to differ significantly. The dose of stimulators giving a maximal primary (I°) response was < 10% the dose of restimulating cells giving a maximal secondary (II°) response, II° responses were further found to be inversely related to the original I° response. This was associated with at least two separate regulatory phenomena. Suppressor cell induction was enhanced at high priming doses while memory cell precursors were preferentially stimulated at very low priming doses. Priming of memory cells could also be demonstrated to occur in the absence of any detectable I° proliferation by utilizing platelets or heat treated stimulators. Memory cells were also a much more resistant than naive cells to both alloantigen induced suppressor cells and to culture activated monocyte suppressor cells. This in vitro model suggests that the human I° and II° responses to alloantigen have both distinct triggering requirements and differential sensitivity to regulatory cells. It is suggested that preferential formation of memory cells under conditions that require no proliferation and which are suboptimal for suppressor cell generation and the acquired resistance of memory cells to down regulation by suppressor cells may contribute to the poor graft prognois of sensitized renal transplant patients.     

Distinct requirements for Ras oncogenesis in human versus mouse cells

The spectrum of tumors associated with oncogenic Ras in humans often differs from those in mice either treated with carcinogens or engineered to sporadically express oncogenic Ras, suggesting that the mechanism of Ras transformation may be different in humans. Ras stimulates primarily three main classes of effector proteins, Rafs, PI3-kinase, and RalGEFs, with Raf generally being the most potent at transforming murine cells. Using oncogenic Ras mutants that activate single effectors as well as constitutively active effectors, we find that the RalGEF, and not the Raf or PI3-kinase pathway, is sufficient for Ras transformation in human cells. Thus, oncogenic Ras may transform murine and human cells by distinct mechanisms, and the RalGEF pathway--previously deemed to play a secondary role in Ras transformation--could represent a new target for anti-cancer therapy.     

Differential effects of a murine and chimeric mouse/human anti-interleukin-2 receptor antibody on human T-cell proliferation.

The preference for interleukin-2 receptor (IL-2R) expression on activated, compared with resting T lymphocytes makes the IL-2R a promising target for selective immunosuppressive therapy. To increase the potential therapeutic effectiveness of anti-IL-2R monoclonals, a chimeric mouse/human variant was constructed from Ig genes isolated from a murine anti-human IL-2R hybridoma cell line, designated AHT54. AHT54 binds to the same or spatially related epitope as IL-2 on the p55 protein that constitutes the low- and high-affinity forms of IL-2R. Although the murine and chimeric AHT54 antibodies inhibited cell-surface binding of IL-2 to the same extent, the chimeric antibodies containing a human IgG1 constant region had substantially more anti-proliferative activity than their murine IgG1 counterparts. Our results indicated that the human constant region of the chimeric antibodies interacted more efficiently than the murine constant region with effector components present in peripheral blood mononuclear cells (PBMC).     

Differential processing of influenza nucleoprotein in human and mouse cells

To investigate how early events in antigen processing affect the repertoire of peptides presented by MHC class I molecules, we compared the presentation of the influenza A nucleoprotein epitope 265 – 273 by HLA-A3 class I molecules in human and mouse cells. Mouse cells that express HLA-A3 failed to present the NP265 – 273 peptide when contained within the full-length nucleoprotein, to HLA-A3-restricted human cytotoxic T lymphocytes. However, when the epitope was generated directly in the cytosol using a recombinant vaccinia virus that expressed the nonamer peptide, mouse cells were recognized by HLA-A3-restricted CTL. Poor transport of the peptide by mouse TAP was not responsible for the defect as co-infection of mouse cells with recombinant vaccinia viruses encoding the full-length nucleoprotein and the human TAP1 and TAP2 peptide transporter complex failed to restore presentation. These results therefore demonstrate a differential processing of the influenza nucleoprotein in mouse and human cells. This polymorphism influences the repertoire of peptides presented by MHC class I molecules at the cell surface.     

Novel domains of expression for orphan receptor tyrosine kinase Ror2 in the human and mouse reproductive system

Background: The noncanonical Wnt receptor and tyrosine kinase Ror2 has been associated with recessive Robinow syndrome (RRS) and dominant brachydactyly type B1. The phenotypes of mouse mutants implicate Ror2 in the development of the heart, lungs, bone, and craniofacial structures, which are affected in RRS. Following a recently identified role of Ror2 in the migration of mouse primordial germ cells, we extensively characterized its expression throughout the fetal internal reproductive system and the postnatal ductal system. Results: We show that Ror2 gene products are present in the germ cells and somatic cells of the testis and the ovary of both the mouse and human fetus. In reproductive tract structures, we find that Ror2 is expressed in the mesonephros, developing Wolffian and Müllerian ducts, and later in their derivatives, the epididymal epithelium and uterine epithelium. Conclusions: This study sets the stage to explore function for this tyrosine kinase receptor in novel regions of expression in the developing reproductive system in both mouse and human. Developmental Dynamics 243:1037–1045, 2014. © 2014 Wiley Periodicals, Inc.     

Structural requirements of the cytoplasmic domains of the human macrophage Fc gamma receptor IIa and B cell Fc gamma receptor IIb2 for the endocytosis of immune complexes.

Two isotypes of the monocyte/macrophage as well as B cell Fc gamma receptor type II (FcRIIa and FcRIIb2, respectively) mainly differ in the length (76 vs. 44 amino acids) and amino acid sequence of their cytoplasmic domains. Only the eight amino acids just behind the putative transmembrane region are identical. Despite this marked difference, both FcRII mediate endocytosis of immune complexes. To determine the functional significance of the cytoplasmic domains, we expressed truncated FcRIIa and FcRIIb2 in FcR- BHK-21 cells. Mutants of both receptors containing only one amino acid (tail-minus) of the cytoplasmic domain failed to mediate immune complex uptake. The significance of the cytoplasmic domain of the receptors could be further demonstrated using a chimeric FcRIII-FcRIIa construct. Therefore we expressed an FcRIII lacking the hydrophobic carboxyl terminus (containing the putative phosphatidyl - inositol - glycan anchor site) fused inframe to the transmembrane and cytoplasmic domain of the FcRIIa in BHK-21 cells. In contrast to the wild type FcRIII, this chimeric receptor mediated immune complex uptake indistinguishable from that mediated by the FcRIIa. Receptor mutants with relatively short cytoplasmic domains (FcRIIb2: 13, and FcRIIa: 16 amino acids) revealed, that these short amino acid stretches are sufficient to allow reduced receptor-mediated endocytosis of bound ligand. Furthermore, using FcRIIa deletion mutants with a cytoplasmic domain consisting of 62, 46, and 28 amino acids, respectively, we found that the capability of these mutants to mediate immune complex uptake decreased gradually with the truncation of the cytoplasmic tails. Thus, only short amino acid sequences of the cytoplasmic domain are sufficient to enable an, albeit reduced, receptor-mediated endocytosis.     

Differential B cell expression of mouse Fc receptor homologs

Five Fc receptor homologs (FcRH1-5) possessing inhibitory and/or activating signaling motifs are differentially expressed during B cell differentiation in humans. In this analysis we describe their three mouse orthologs, moFcRH1, moFcRH2 and moFcRH3. The moFcRH genes are located in a chromosome 3 region that is syntenic with the FcRH locus on human chromosome 1. They encode proteins with 2-5 Ig-like domains that share 20-61% extracellular identity with their human counterparts. One moFcRH1 isoform lacks a transmembrane domain as do both moFcRH2 isoforms. The other moFcRH1 isoform and two moFcRH3 isoforms have transmembrane domains and cytoplasmic ITIM and ITAM-like consensus sequences implying their inhibitory or activating signaling potential. Whereas the moFcRH1 and moFcRH3 orthologs are preferentially expressed at different stages in B cell differentiation, the structurally novel moFcRH2 gene is expressed in non-lymphoid tissues. The highly restricted pattern of moFcRH3 expression suggests this member of the phylogenetically conserved FcRH family may have an important immunoregulatory role in marginal zone B cells.     

Differential requirements for hematopoietic commitment between human and rhesus embryonic stem cells

Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs. To accomplish this goal, we compared the hematopoietic differentiation of hESCs with that of rESCs in both EB culture and stroma coculture. Initially, undifferentiated rESCs and hESCs were adapted to growth on Matrigel without a change in their phenotype or karyotype. Subsequent differentiation of rESCs in OP9 stroma led to the development of CD34(+)CD45(-) cells that gave rise to endothelial cell networks in methylcellulose culture. In the same conditions, hESCs exhibited convincing hematopoietic differentiation. In cytokine-supplemented EB culture, rESCs demonstrated improved hematopoietic differentiation with higher levels of CD34(+) and detectable levels of CD45(+) cells. However, these levels remained dramatically lower than those for hESCs in identical culture conditions. Subsequent plating of cytokine-supplemented rhesus EBs in methylcellulose culture led to the formation of mixed colonies of erythroid, myeloid, and endothelial cells, confirming the existence of bipotential hematoendothelial progenitors in the cytokine-supplemented EB cultures. Evaluation of four different rESC lines confirmed the validity of these disparities. Although rESCs have the potential for hematopoietic differentiation, they exhibit a pause at the hemangioblast stage of hematopoietic development in culture conditions developed for hESCs.     

Activation of cAMP-dependent protein kinase is necessary for actin rearrangements in human neutrophils during phagocytosis

We have investigated the role of cAMP and cAMP-dependent protein kinase (cAPK) in neutrophil phagocytosis. Inhibition of cAPK with H-89 reduced complement- and IgG-dependent phagocytosis to 83 and 46%, respectively. Fluorescence intensity measurements of phalloidin-stained actin in neutrophils showed a reduced amount of filamentous actin (F-actin) in pseudopods and around the phagosome in cells treated with H-89 or cAMP-elevating agents (forskolin and rolipram). The amount of phosphotyrosine-containing proteins was also reduced in pseudopods and around the phagosome. Taken together, the data show that cAMP/cAPK regulates F-actin reorganization during receptor-mediated phagocytosis, particularly triggered by IgG-FcR interaction. Our results support the hypothesis that active subcortical reorganization of F-actin is a prerequisite for FcR-mediated phagocytosis, but is less important during CR3-mediated ingestion.     

酪氨酸激酶受体KIT在小鼠结肠黏膜上皮的表达及其功能

目的 探讨酪氨酸激酶受体KIT在小鼠结肠黏膜上皮的表达及作用。 方法 应用Western blotting和RT-PCR技术检测c-kit基因及KIT
蛋白在野生型C57BL/6小鼠结肠黏膜上皮的表达;用免疫荧光染色显示野生型C57BL/6小鼠以及乙基亚硝酸钠（ENU）诱变的c-kit基因点突变纯
合子小鼠（Wads ^m/m）结肠黏膜上皮KIT阳性细胞的部位;腹腔注射5-溴脱氧尿嘧啶核苷（BrdU）（30 mg/kg）观察对比野生型以及Wads m/m
小鼠结肠黏膜上皮BrdU掺入情况及动态变化,分析KIT在结肠黏膜上皮更新过程的作用。 结果 RT-PCR检测结果表明,在野生型小鼠结肠黏膜组
织表达 c-kit基因, Western blotting证明在肠黏膜组织存在KIT蛋白;免疫荧光染色显示,KIT阳性细胞位于结肠黏膜上皮,主要位于肠腺隐
窝基底部,但是Wads ^m/m小鼠KIT阳性细胞数以及KIT蛋白表达量较野生型小鼠明显减少;BrdU掺入实验发现,Wads ^m/m小鼠远端结肠黏膜上皮
BrdU摄入和更新速度较野生型小鼠明显减慢。结论 结肠黏膜上皮表达KIT蛋白与结肠黏膜上皮的更新密切相关。     

Differential expression of Toll-like receptor 2 in human cells

Human Toll-like receptor 2 (TLR2) is a receptor for a variety of microbial products and mediates activation signals in cells of the innate immune system. We have investigated expression and regulation of the TLR2 protein in human blood cells and tissues by using two anti-TLR2 mAbs. Only myelomonocytic cell lines expressed surface TLR2. In tonsils, lymph nodes, and appendices, activated B-cells in germinal centers expressed TLR2. In human blood, CD14+ monocytes expressed the highest level of TLR2 followed by CD15+ granulocytes, and CD19+ B-cells, CD3+ T-cells, and CD56+ NK cells did not express TLR2. The level of TLR2 on monocytes was after 20 h up-regulated by LPS, GM-CSF, IL-1, and IL-10 and down-regulated by IL-4, IFN-gamma, and TNF. On purified granulocytes, LPS, GM-CSF, and TNF down-regulated, and IL-10 modestly increased TLR2 expression after 2 h. These data suggest that TLR2 protein expression in innate immune cells is differentially regulated by inflammatory mediators.     

Differential activation of human and mouse Toll-like receptor 4 by the adjuvant candidate LpxL1 of Neisseria meningitidis

Neisseria meningitidis LpxL1 lipopolysaccharide (LPS) bearing penta-acylated lipid A is considered a promising adjuvant candidate for inclusion in future N. meningitidis vaccines, as it elicits a markedly reduced endotoxic response in human macrophages relative to that in wild-type (hexa-acylated) LPS, while it is an equally effective adjuvant in mice. As dendritic cells (DC) and Toll-like receptors (TLR) are regarded as central mediators in the initiation of an immune response, here we evaluated the ability of LpxL1 LPS to mature and to activate human DC and examined its TLR4-/MD-2-activating properties. Unexpectedly, purified LpxL1 LPS displayed minimal human DC-stimulating properties compared to wild-type LPS. Although whole bacteria induced DC maturation and activation irrespective of their type of LPS, the LpxL1 mutant failed to activate the human recombinant TLR4/MD-2 complex expressed in HeLa cells. Similarly, purified LpxL1 LPS was unable to activate human TLR4/MD-2 and it even acted as an antagonist of wild-type LPS. Both wild-type and LpxL1 LPSs activated the murine TLR4/MD-2 complex, consistent with their abilities to induce maturation and activation of murine DC. Assays with cells transfected with different combinations of human and murine TLR4 and MD-2 indicated that TLR4 was a more-major determinant of the LPS response than MD-2. The species-specific activation of the TLR4/MD-2 complex by LpxL1 LPS may have an impact on the use of LpxL1 LPS as an adjuvant and the use of murine immunization models in human meningococcal vaccine development.     

Receptor-mediated endocytosis of glucagon in isolated mouse hepatocytes

The binding of glucagon to the cell surface and the pathway of intracellular transport of the hormone in isolated mouse hepatocytes were studied by autoradiography, colloidal gold-labeled glucagon (Au-glucagon), and biochemical methods. In cells incubated with 1251-glucagon at 4 degrees C, the label was mainly localized to the plasma membrane even after 60 min of incubation. At 20 degrees C, the labeled ligand was internalized by the cells and the amount of internalized ligand increased with time of incubation. At 37 degrees C, the ligand was rapidly internalized and found to be associated with coated or uncoated vesicles. Au-glucagon experiments revealed clearly the process of internalization of glucagon. Au-glucagon bound to the plasma membrane was transported to coated regions and then internalized into vesicles via coated pits. Biochemical results supported these findings from autoradiography and Au-glucagon experiments. Thus, glucagon is internalized by hepatocytes via receptor-mediated endocytosis.