Identification of early postmitotic cells in distinct embryonic sites and their possible roles in morphogenesis

Differential proliferation within defined embryonic anlage is likely to play a major role in morphogenesis. We have identified cell populations in the avian embryo that begin exiting the cell cycle as early as the 25-somite stage. These include first the floor plate and then the roof plate of the neural tube, cells that constitute the lamina terminalis and the diencephalic-mesencephalic junction of the developing brain. Outside the nervous system, the central portion of the notochord contains early postmitotic cells. In the heart, such cells will populate the epimyocardium at the level of the truncus arteriosus exclusively and the endocardial cushions that serve as an anchor for the growing intracardial septa. Surprisingly, the endoderm at the level of the prospective midgut is composed of post-mitotic progenitors. These cells are later found both in the caudal portion of the duodenum and in derivatives adjacent to the umbilical region of the primitive midgut. The possible implications of this early, localized withdrawal from the cell cycle to morphogenetic events and lineage segregation are discussed.     

Connecting ecology to daily life: how students and teachers relate food webs to the food they eat

This study used sociocultural learning theory to better understand how middle and high school environmental science and biology students and pre- and in-service science teachers connect the daily life activity of eating to the food web model learned in school. We sought to understand how student and teacher perceptions of the environment and their experiences influenced their responses to interview questions regarding this topic. Findings, based on transcribed interviews with 54 study participants, indicate that three quarters of teachers and students were unable to connect the food they eat with ecosystem food webs. Even so, many respondents particularly those from elite public schools, did not demonstrate common food web misconceptions identified by other researchers, instead showing a sophisticated understanding of food web interactions. These findings indicate that even though participants were proficient in their school science understanding of food web interactions, they did not readily think about how their everyday out of school activities, like eating, relate to those interactions. This may be representative of a more general disconnect between formal ecology instruction and daily life activities. We provide several recommendations for how this disconnect can be remedied in our classrooms.     

Pericytes in human breast stroma and the cells to which they relate.

The function of pericytes, the cells nearest the microvascular endothelium, has long been debated. On the basis of ultrastructural studies it is pointed out that they have specialised features in common with endothelial cells of the lymphatic labyrinth of the human breast. The latter are in continuity with the initial lymphatics. These features, which include points of stromal attachment, allow contraction or relaxation of the cell-processes. The cytoplasmic processes of the pericyte, when relaxed, form areas of contact with the blood vascular endothelium. Subsequent contraction may lead to loss of contact and increase in the area of vascular endothelium available for diffusion. The pericyte is thus equipped to act as a regulatory link between the blood vascular endothelium and that of the fine lymphatic system.     

The complexity of the CD4 T-cell responses: old and new T-cell subsets

The T-cell compartment of the immune system reacts to an enormous variety of antigens, including self antigens, due to its a wide repertoire of T-cell clones. Self-reactive T cells undergo a negative selection process resulting in apoptosis of T cells with high affinity for self-peptides. Self-reactive T cells escaped to negative selection are then controlled by natural T regulatory (Treg) cells. Regulation also controls excessive effector T-cell responses. Three types of effector T cells are recognized: T helper 1 (Th1) cells, which protect against intracellular bacteria; Th2 cells, which play a role against parasites; Th17 cells, which would face extracellular bacteria, but also are involved in autoimmunity. Effector T-cell polarization is determined by the complex interaction of antigen-presenting cells with naive T cells and involves a multitude of factors, including the dominant cytokine environment, costimulatory molecules, type and load of antigen presented and signaling cascades. The decision for the immune response to go in a certain direction is based not onto one signal alone, rather onto many different elements acting synergistically, antagonistically and through feedback loops leading to activation of Th1, Th2, or Th17 responses. Both Th1 and Th2 can be suppressed by adaptive Treg cells through contact-dependent mechanisms and/or cytokines.     

Susceptibility of human T cells, T-cell subsets, and B cells to cryopreservation

We have studied the number of viable and functionally active T and B lymphocytes obtainable after cryopreservation to determine the best and most practical way to recover the maximal number of viable and functionally active cells. Assays were done on purified populations of human T and B cells recovered after cryopreservation. The results were compared to those obtained from similar types of cells fractionated from fresh and from cryopreserved mononuclear cells. The number of viable T cells recovered after cryopreservation was significantly lower than the number of viable T cells obtained from either fresh or cryopreserved mononuclear cells. The residual viable T cells recovered after cryopreservation showed significantly reduced blastogenic activity in response to pokeweed mitogen (PWM) stimulation. This occurred despite their normal blastogenic response to phytohemagglutinin and their normal ability to help B cells in the production of immunoglobulins following PWM stimulation. The reduction in the blastogenic responses of these T cells to PWM stimulation is attributed to the loss of a portion of the PWM responding subset of T cells. The loss in this subset of T cells was related to the exposure of cells to ammonium chloride prior to cryopreservation. The viability and functional abilities of B cells were not affected regardless of whether purification was done before or after cryopreservation. These findings indicate that extrinsic membrane damage to T cells induced prior to cryopreservation can affect the viability and responsiveness of a certain population of normal T cells. The damage can be minimized by reversing the sequence of T-cell isolation and freezing so that isolation of T cells is done after, rather than before, freezing. These results could be important in the study of T cells from patients with T-cell abnormalities, since the patients' cells could have an intrinsic membrane defect which would make them sensitive to freezing similar to that induced by extrinsic damage.     

DC-SIGN and CD150 have distinct roles in transmission of measles virus from dendritic cells to T-lymphocytes

Measles virus (MV) is among the most infectious viruses that affect humans and is transmitted via the respiratory route. In macaques, MV primarily infects lymphocytes and dendritic cells (DCs). Little is known about the initial target cell for MV infection. Since DCs bridge the peripheral mucosal tissues with lymphoid tissues, we hypothesize that DCs are the initial target cells that capture MV in the respiratory tract and transport the virus to the lymphoid tissues where MV is transmitted to lymphocytes. Recently, we have demonstrated that the C-type lectin DC-SIGN interacts with MV and enhances infection of DCs in cis. Using immunofluorescence microscopy, we demonstrate that DC-SIGN+ DCs are abundantly present just below the epithelia of the respiratory tract. DC-SIGN+ DCs efficiently present MV-derived antigens to CD4+ T-lymphocytes after antigen uptake via either CD150 or DC-SIGN in vitro. However, DC-SIGN+ DCs also mediate transmission of MV to CD4+ and CD8+ T-lymphocytes. We distinguished two different transmission routes that were either dependent or independent on direct DC infection. DC-SIGN and CD150 are both involved in direct DC infection and subsequent transmission of de novo synthesized virus. However, DC-SIGN, but not CD150, mediates trans-infection of MV to T-lymphocytes independent of DC infection. Together these data suggest a prominent role for DCs during the initiation, dissemination, and clearance of MV infection.     

BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood

We have generated a panel of mAbs that identify three presumably novel human dendritic cell Ags: BDCA-2, BDCA-3, and BDCA-4. In blood, BDCA-2 and BDCA-4 are expressed on CD11c(-) CD123(bright) plasmacytoid dendritic cells, whereas BDCA-3 is expressed on small population of CD11c(+) CD123(-) dendritic cells. All three Ags are not detectable on a third blood dendritic cell population, which is CD1c(+) CD11c(bright) CD123(dim), or on any other cells in blood. BDCA-4 is also expressed on monocyte-derived and CD34(+) cell-derived dendritic cells. Expression of all three Ags dramatically changes once blood dendritic cells undergo in vitro maturation. BDCA-2 is completely down-regulated on plasmacytoid CD11c(-) CD123(bright) dendritic cells, expression of BDCA-3 is up-regulated on both plasmacytoid CD11c(-) CD123(bright) dendritic cells and CD1c(+) CD11c(bright) CD123(dim) dendritic cells, and expression of BDCA-4 is up-regulated on CD1c(+) CD11c(bright) CD123(dim) dendritic cells. BDCA-2 is rapidly internalized at 37 degrees C after mAb labeling. The three presumably novel Ags serve as specific markers for the respective subpopulations of blood dendritic cells in fresh blood and will be of great value for their further analysis and to evaluate their therapeutic potential.     

Further characterization of mitogen-induced autorosette-forming cells: correlation with T-cell subsets and with lymphocyte proliferative responses to mitogens

Spontaneous autologous rosette-forming cells (ARFC), which form rosettes with autologous erythrocytes, have been of interest as a subset of thymus-derived lymphocytes (T cells). An association of these cells with concanavalin A (Con A)-induced ARFC has been suggested. Furthermore, the Con A-induced ARFC have been shown to be a suppressor T-cell subset in the Con A-generated suppressor system. We have previously reported the induction of ARFC from T cells by several T-cell mitogens such as phytohemagglutinin-P (PHA) and allogeneic non-T cells other than Con A. In the present report, we further characterized the mitogen-induced ARFC and have extended the study to patients with systemic lupus erythematosus (SLE). We have found that ARFC are also inducible from peripheral blood T cells by pokeweed mitogen (PWM). Studies of T-cell surface markers on the ARFC using OKT monoclonal antibodies confirmed the induction of ARFC from both OKT4- and OKT8-reactive T cells by either Con A, PHA, or PWM stimulation. However, OKT4-reactive T cells were the major cellular source of the ARFC induced by all of the mitogens. In studies of SLE patients, proportions of both Con A- and PWM-induced ARFC were found to be significantly low in PBL of SLE patients treated with moderate or large doses of prednisone, with or without concomitant immunosuppressants, but not in SLE patients without such treatment. Proportional analysis of the T cells and their subsets suggested association of these alterations in the mitogen-induced ARFC with the OKT4-reactive T cells, since a significant decrease in the OKT4-reactive T-cell subset was demonstrated in the PBL of these patients. Proportions of PHA-induced ARFC, however, were not significantly different between SLE patients and healthy adults. Moreover, positive correlations of the mitogen-induced ARFC with lymphocyte proliferative responses to each mitogen were established in both SLE patients and healthy adults. These results further support our previous observation that suggest the receptors for autologous erythrocytes are enhanced or reexpressed on those T cells which are highly activated by mitogens.     

Differential regulation of formyl peptide receptor-like 1 expression during the differentiation of monocytes to dendritic cells and macrophages

Monocytes are the common precursors for myeloid dendritic cells (DC) and macrophages. Identification of chemotactic receptors expressed by myeloid DC, macrophages, and their precursors in the course of differentiation and maturation is important not only for elucidation of their in vivo trafficking, but also for understanding of the functional distinction between DC and macrophages. We chose to study formyl peptide receptor like-1 (FPRL1), a chemotactic receptor known to interact with several endogenous agonists that are involved in inflammatory and host defense responses. Here we show that FPRL1 is down-regulated as monocytes differentiate into DC. This down-regulation occurs at both mRNA and functional levels. Therefore, the interaction of FPRL1 with its agonists is more likely to regulate the in vivo trafficking of DC precursors than DC. In contrast, FPRL1 expression is maintained at both mRNA and functional levels as monocytes differentiate into macrophages. Thus, our results demonstrate further distinctions between myeloid DC and macrophages, albeit they share a common precursor. The fact that macrophages rather than myeloid DC express functional FPRL1 suggests that this chemotactic receptor may be more involved in inflammatory reactions and innate host defense than in adaptive immune responses.     

Coordinated and distinct roles for IFN-alpha beta,IL-12, and IL-15 regulation of NK cell responses to viral infection

NK cell cytotoxicity, IFN-gamma expression, proliferation, and accumulation are rapidly induced after murine CMV infections. Under these conditions, the responses were shown to be elicited in overlapping populations. Nevertheless, there were distinct signaling molecule requirements for induction of functions within the subsets. IL-12/STAT4 was critical for NK cell IFN-gamma expression, whereas IFN-alphabeta/STAT1 were required for induction of cytotoxicity. The accumulation/survival of proliferating NK cells was STAT4-independent but required IFN-alphabeta/STAT1 induction of IL-15. Taken together, the results define the coordinated interactions between the cytokines IFN-alphabeta, IL-12, and IL-15 for activation of protective NK cell responses during viral infections, and emphasize these factors' nonredundant functions under in vivo physiological conditions.     

Gene expression analysis in human monocytes, monocyte-derived dendritic cells, and alpha-galactosylceramide-pulsed monocyte-derived dendritic cells.

In vitro proliferation and functional activation of V alpha 24NKT cells following stimulation with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with alpha-GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with alpha-GalCer (alpha-GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in alpha-GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, alpha-GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with alpha-GalCer, suggesting a novel immunomodulating effect of alpha-GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNF alpha for two extra days.     

Functional subsets of human monocytes defined by monoclonal antibodies: a distinct subset of monocytes contains the cells capable of inducing the autologous mixed lymphocyte culture

The induction of most immune responses requires the close cooperation between T cells and antigen-presenting cells (APC), presumably of monocyte/macrophage (M phi) lineage. To characterize human APC further, we used two monoclonal antibodies, OKM1 and OKM5, to isolate and identify M phi subsets. OKM1 has been described and recognizes cell surface antigens on most M phi and granulocytes. OKM5 recognizes cell surface determinants present on the majority of human M phi but does not recognize other hematopoietic cell types. A small subset of peripheral blood M phi is OKM1-OKM5+. Human peripheral blood E- cells were separated into OKM1+ and OKM1- subsets by a rosetting technique utilizing anti-Ig-coated red cells. The capacity to present self antigens in the autologous mixed lymphocyte culture (AMLC) resided predominantly within the E-OKM1- subset, even if surface membrane Ig-positive cells were eliminated. Similar experiments showed that the ability to stimulate in AMLC was contained in the E-OKM5+ population and in fact resided primarily within the E-OKM1-OKM5+ subset. All of these subsets were able to trigger allogeneic T cells to proliferate. The capacity of these APC subsets to present soluble antigens (mumps, tetanus toxoid) was also examined. The data demonstrated that although the majority of these APC are E-OKM1+, E-OKM1-OKM5+ cells can also present foreign antigen. Taken together, these data suggest OKM1 and OKM5 can be used to isolate two functionally distinct human M phi subsets. One subset (E-OKM1+) is capable of presenting soluble antigens but shows minimal ability to trigger AMLC. The other subset (E-OKM1-OKM5+) can also present soluble antigens but is the predominant subset that can trigger AMLC.     

Relationships between distinct blood monocyte subsets and migrating intestinal lymph dendritic cells in vivo under steady-state conditions

The origins of dendritic cells (DCs) are poorly understood. In inflammation, DCs can arise from blood monocytes (M(O)s), but their steady-state origin may differ, as shown for Langerhans cells. Two main subsets of M(O)s, defined by expression of different chemokine receptors, CCR2 and CX(3)CR1, have been described in mice and humans. Recent studies have identified the inflammatory function of CCR2(high)CX(3)CR1(low) M(O)s but have not defined unambiguously the origin and fate of CCR2(low)CX(3)CR1(high) cells. In this study, we show that rat M(O)s can also be divided into CCR2(high)CX(3)CR1(low)(CD43(low)) and CCR2(low)CX(3)CR1(high)(CD43(high)) subsets with distinct migratory properties in vivo. Using whole body perfusion to obtain M(O)s, including the marginating pool, we show by adoptive transfer that CD43(low) M(O)s can differentiate into CD43(high) M(O)s in blood without cell division. By adoptive transfer of blood M(O)s followed by collection of pseudoafferent lymph, we show for the first time that a small proportion of intestinal lymph DCs are derived from CCR2(low)CX(3)CR1(high)(CD43(high)) blood M(O)s in vivo under steady-state conditions. This study confirms one of the possible origins of CCR2(low)CX(3)CR1(high) blood M(O)s and indicate that they may contribute to migratory intestinal DCs in vivo in the absence of inflammatory stimuli.     

The roles of Toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNAs) can function as powerful immune adjuvants by activating APC. Compared with conventional phosphorothioate-backbone CpG DNAs, another type of CpG DNAs, called an A or D type (A/D-type), possesses higher ability to induce IFN-alpha production. Conventional CpG DNAs can exert their activity through Toll-like receptor 9 (TLR9) signaling, which depends on a cytoplasmic adapter, MyD88. However, it remains unknown how A/D-type CpG DNAs exhibit their immunostimulatory function. In this study we have investigated murine dendritic cell (DC) responses to these two distinct CpG DNAs. Not only splenic, but also in vitro bone marrow-derived, DCs could produce larger amounts of IFN-alpha in response to A/D-type CpG DNAs compared with conventional CpG DNAs. This IFN-alpha production was mainly due to the B220(+) DC subset. On the other hand, the B220(-) DC subset responded similarly to both CpG DNAs in terms of costimulatory molecule up-regulation and IL-12 induction. IFN-alpha, but not IL-12, induction was dependent on type I IFN. However, all activities of both CpG DNAs were abolished in TLR9- and MyD88-, but were retained in DNA-PKcs-deficient DCs. This study demonstrates that the TLR9-MyD88 signaling pathway is essential for all DC responses to both types of CpG DNAs.     

A novel dendritic cell-induced model of erosive inflammatory arthritis: distinct roles for dendritic cells in T cell activation and induction of local inflammation

Transferring collagen-pulsed, bone marrow-derived dendritic cells (DCs) into congenic DBA/1 recipient mice produced arthritis in joints adjacent to the site of DC transfer and could be inhibited by treatment with TNF antagonists. Disease was Ag specific, as transfer of control, unpulsed DCs, or DCs pulsed with OVA did not produce arthritis. In contrast to other experimental arthritis models, DC-induced arthritis localized to the site of injection and did not spontaneously generalize to uninvolved joints, despite the demonstration of circulating collagen-reactive T cells. Similarly, transfer of T cells primed by collagen/DCs was not sufficient to produce arthritis in recipient mice. In collagen/DC-primed mice however, disease could be induced in uninvolved joints by local administration of noncollagen-pulsed DCs and this could be reduced through TNF inhibition. Similarly, injection of collagen/DC-primed mice with low-dose TNF also resulted in local induction of arthritis, as did administration of TNF to mice receiving T cells from collagen/DC but not OVA/DC-primed mice. Thus, we have demonstrated for the first time that administration of collagen-pulsed mature DCs is sufficient for the induction of arthritis. Furthermore, this disease process is mediated through both adaptive and innate effects of DCs; first, priming of autoreactive T cells and, second, induction of local inflammation via mediators such as TNF.     

Differential expression of co-signal molecules and migratory properties in four distinct subsets of migratory dendritic cells from the oral mucosa

Variations in co-signal ligand expression and cytokine production greatly influence the antigen-presenting properties of migrating DCs in regional lymph nodes (RLNs). Here we investigated DCs migrating from the oral mucosa using CD326 and CD103 antigens for discriminate CD207⁺ Langerhans cells (LCs) from CD207⁺ submucosal DCs (SMDCs). Similar to DCs migrating from the skin, we identified four distinct oral mucosal DC (OMDC) subsets, CD11c^hiCD207⁻CD103⁻CD326^intCD11b^hi (F1; resident CD11b^hi SMDCs), CD11c^int/loCD207^-CD103^-CD326^loCD11b^int/hi (F2; newly recruited blood-derived SMDCs), CD11c^int/loCD207⁺CD103⁺CD326^int/hiCD11b^lo (CD103⁺ F3; resident CD207⁺ SMDCs), and CD11c^int/loCD207⁺CD103^-CD326^int/hiCD11b^lo (CD103^- F3; resident LCs). F1 DCs migrated rapidly after fluorescein isothiocyanate (FITC) painting and expressed notably high levels of CD86, CD273, and CD274 at an earlier time point. In contrast, CD103⁻ LCs expressing the highest levels of the epithelial cell adhesion molecule CD326 accounted for a minor subset at the earlier time point, but increased slowly with CD103⁺CD207⁺ SMDCs. However, their expression of CD86, CD273, and CD274 was very limited. The delayed migration and limited induction of co-signal ligands suggest that roles of OMLCs are distinct from those of the other three DC subsets. The identification of distinct subsets of OMDCs in RLNs may benefit efforts to determine the functional specialization of each subset in T cell responses against orally administrated antigens.     

Maturation of circulating dendritic cells and imbalance of T-cell subsets in patients with squamous cell carcinoma of the head and neck

Interactions between dendritic cells (DCs) and T cells play a pivotal role in the regulation and maintenance of immune responses. In cancer patients, various immunological abnormalities have been observed in these immune cells. Here, we investigated proportions and the phenotype of DCs and the cytokine profile of T-cell subsets in the peripheral blood of patients with squamous cell carcinoma of the head and neck (SCCHN), using multicolor flow cytometry. The percentage of myeloid (CD11c+), but not plasmacytoid (CD123+) DCs, was significantly lower (P<0.05) and expression of HLA-DR was significantly decreased in total and myeloid DCs of cancer patients compared to healthy donors. Simultaneous analyses of T-cell subsets in the patients’ circulation showed significantly increased proportions of CD4+ T cells expressing Th1 and Th2 cytokines after ex vivo stimulation without any skewing in the Th1/Th2 ratio. The relative level of HLA-DR expression on myeloid or total DCs positively correlated with the Th1/Th2 ratio (P<0.01), and the proportion of total circulating DCs was inversely correlated with that of regulatory CD4+CD25+ T cells (P<0.01). These results suggest that the decreased proportion of circulating DCs and decreased HLA-DR expression in DCs may have a major impact on systemic immune responses in patients with SCCHN.