Stromal cell-derived factor-1-CXC chemokine receptor 4 interactions play a central role in CD4+ T cell accumulation in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is characterized by the accumulation of CD4(+) memory T cells in the inflamed synovium. To address the mechanism, we analyzed chemokine receptor expression and found that the frequency of CXC chemokine receptor (CXCR)4 expressing synovial tissue CD4(+) memory T cells was significantly elevated. CXCR4 expression could be enhanced by IL-15, whereas stromal cell-derived factor (SDF)-1, the ligand of CXCR4, was expressed in the RA synovium and could be increased by CD40 stimulation. SDF-1 stimulated migration of rheumatoid synovial T cells and also inhibited activation-induced apoptosis of T cells. These results indicate that SDF-1-CXCR4 interactions play important roles in CD4(+) memory T cell accumulation in the RA synovium, and emphasize the role of stromal cells in regulating rheumatoid inflammation.     

IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells

IL-21 is a type I cytokine that influences the function of T cells, NK cells, and B cells. In this study, we report that IL-21 plays a major role in stimulating the differentiation of human B cells. When human B cells were stimulated through the BCR, IL-21 induced minimal proliferation, IgD down-modulation, and small numbers of plasma cells. In contrast, after CD40 engagement, IL-21 induced extensive proliferation, class switch recombination (CSR), and plasma cell differentiation. Upon cross-linking both BCR and CD40, IL-21 induced the largest numbers of plasma cells. IL-21 drove both postswitch memory cells as well as poorly responsive naive cord blood B cells to differentiate into plasma cells. The effect of IL-21 was more potent than the combination of IL-2 and IL-10, especially when responsiveness of cord blood B cells was examined. IL-21 costimulation potently induced the expression of both B lymphocyte-induced maturation protein-1 (BLIMP-1) and activation-induced cytidine deaminase as well as the production of large amounts of IgG from B cells. Despite the induction of activation-induced cytidine deaminase and CSR, IL-21 did not induce somatic hypermutation. Finally, IL-2 enhanced the effects of IL-21, whereas IL-4 inhibited IL-21-induced plasma cell differentiation. Taken together, our data show that IL-21 plays a central role in CSR and plasma cell differentiation during T cell-dependent B cell responses.     

The excitoprotective effect of N-methyl-D-aspartate receptors is mediated by a brain-derived neurotrophic factor autocrine loop in cultured hippocampal neurons

The neuroprotective effect and molecular mechanisms underlying preconditioning with N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons have not been described. Pre-incubation with subtoxic concentrations of the endogenous neurotransmitter glutamate protects vulnerable neurons against NMDA receptor-mediated excitotoxicity. As a result of physiological preconditioning, NMDA significantly antagonizes the neurotoxicity resulting from subsequent exposure to an excitotoxic concentration of glutamate. The protective effect of glutamate or NMDA is time- and concentration-dependent, suggesting that sufficient agonist and time are required to establish an intracellular neuroprotective state. In these cells, the TrkB ligand, brain-derived neurotrophic factor (BDNF) attenuates glutamate toxicity. Therefore, we tested the hypothesis that NMDA protects neurons via a BDNF-dependent mechanism. Exposure of hippocampal cultures to a neuroprotective concentration of NMDA (50 microM) evoked the release of BDNF within 2 min without attendant changes in BDNF protein or gene expression. The accumulated increase of BDNF in the medium is followed by an increase in the phosphorylation (activation) of TrkB receptors and a later increase in exon 4-specific BDNF mRNA. The neuroprotective effect of NMDA was attenuated by pre-incubation with a BDNF-blocking antibody and TrkB-IgG, a fusion protein known to inhibit the activity of extracellular BDNF, suggesting that BDNF plays a major role in NMDA-mediated survival. These results demonstrate that low level stimulation of NMDA receptors protect neurons against glutamate excitotoxicity via a BDNF autocrine loop in hippocampal neurons and suggest that activation of neurotrophin signaling pathways plays a key role in the neuroprotection of NMDA.     

Genomics and variation of ionotropic glutamate receptors: implications for neuroplasticity

Summary. We used two approaches to identify sequence variants in ionotropic glutamate receptor (IGR) genes: high-throughput screening and resequencing techniques, and information mining of public (e.g. dbSNP, ENSEMBL) and private (i.e. Celera Discovery System) sequence databases. Each of the 16 known IGRs is represented in these databases, their positions on a canonical physical map are established. Comparisons of mouse, rat, and human sequences revealed substantial conservation among these genes, which are located on different chromosomes but found within syntenic groups of genes. The IGRs are members of a phylogenetically ancient gene family, sharing similarities with glutamate-like receptors in plants. Parsimony analysis of amino acid sequences groups the IGRs into three distinct clades based on ligand-binding specificity and structural features, such as the channel pore and membrane spanning domains. A collection of 38 variants with amino acid changes was obtained by combining screening, resequencing, and informatics approaches for several of the IGR genes. This represents only a fraction of the sequence variation across these genes, but in fact these may constitute a large fraction of the common polymorphisms at these genes and these polymorphisms are a starting point for understanding the role of these variants in function.Genetically influenced human neurobehavioral phenotypes are likely to be linked to IGR genetic variants. Because ionotropic glutamate receptor activation leads to calcium entry, which is fundamental in brain development and in forms of synaptic plasticity essential for learning and memory and is essential for neuronal survival, it is likely that sequence variants in IGR genes may have profound functional roles in neuronal activation and survival mechanisms.     

Staphylococcal protein a deletes B-1a and marginal zone B lymphocytes expressing human immunoglobulins: an immune evasion mechanism

Protein A (SpA) of Staphylococcus aureus is endowed with the capacity to interact with the H chain variable region (V(H)) of human Abs and to target >40% of B lymphocytes. To investigate whether this property represents a virulence factor and to determine the in vivo consequences of the confrontation of SpA with B lymphocytes, we used transgenic mice expressing fully human Abs. We found that administration of soluble SpA reduces B-1a lymphocytes of the peritoneal cavity and marginal zone B lymphocytes of the spleen, resulting in a markedly deficient type 2 humoral response. Single-cell PCR analysis and sequencing of the Ab V(H) gene repertoire revealed a significant reduction of V(H)3+ marginal zone B cells. Since the two B lymphocyte subsets targeted are involved in innate immune functions, our data suggest that crippling of humoral immunity by S. aureus represents an immune evasion mechanism that may aggravate recurrent infections.     

Co-activation of the phosphatidylinositol-3-kinase/Akt signaling pathway by N-methyl-D-aspartate and TrkB receptors in cerebellar granule cell neurons

Summary.  Neuroprotective concentrations of N-methyl-D-aspartate (NMDA) promote survival of cerebellar granule cell neurons against glutamate excitotoxicity through a TrkB receptor-mediated brain-derived neurotrophic factor (BDNF) autocrine loop. However, the intracellular signaling pathway(s) are not clear. Our results show that PI-3 kinase/Akt is activated by either NMDA or BDNF displaying differential kinetics. BDNF and NMDA increased Akt phosphorylation within 5 minutes but maximal activation by NMDA was observed at 3 hours. Akt phosphorylation was completely blocked by the PI-3 kinase inhibitor LY294002. NMDA-mediated activation of Akt was completely blocked by MK-801 and partially blocked by the TrkB receptor inhibitor, K252a, indicating the requirement of TrkB receptors for maximal activation by NMDA. In contrast, BDNF-induced Akt phosphorylation was abolished by K252a, but not by the addition of MK-801. Therefore, the PI-3 kinase/Akt pathway is co-activated by NMDA and TrkB receptors. The kinetics of BDNF and NMDA-mediated activation of PI-3 kinase/Akt suggests that they have different roles in intraneuronal time-related events. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Enhancement of antigen- and mitogen-induced human T lymphocyte proliferation by tumor necrosis factor-alpha

The capacity of human recombinant tumor necrosis factor-alpha (rTNF alpha) to modulate human T cell proliferation was examined. To examine the effect of rTNF alpha on the responding T cell directly, T cell activation was studied in the absence of viable accessory cells (AC). Highly purified AC-depleted peripheral blood T4 or T8 cells were stimulated with immobilized monoclonal antibodies to the cluster of differentiation (CD)3 molecular complex, an AC-independent stimulus. rTNF alpha augmented anti-CD3-stimulated T4 and T8 cell proliferation. The capacity of rTNF alpha to enhance T cell proliferation varied inversely with the density of immobilized anti-CD3 and the number of responding cells in each culture. The capacity of rTNF alpha to enhance antigen-induced T4 cell proliferation was also examined. Antigen-bearing paraformaldehyde-fixed antigen-presenting cells induced modest T4 cell proliferation when cultured in flat-bottomed wells; this response was enhanced by rTNF alpha. The results demonstrate that rTNF alpha has direct effects on T cells, facilitating their capacity to proliferate in response to mitogens and antigens. These data indicate that rTNF alpha may play an immunoregulatory role, enhancing the proliferation of T lymphocytes.     

B cells in autoimmunity

B-cell development is tightly regulated, including the induction of B-cell memory and antibody-secreting plasmablasts and plasma cells. In the last decade, we have expanded our understanding of effector functions of B cells as well as their roles in human autoimmune diseases. The current review addresses the role of certain stages of B-cell development as well as plasmablasts/plasma cells in immune regulation under normal and autoimmune conditions with particular emphasis on systemic lupus erythematosus. Based on preclinical and clinical data, B cells have emerged increasingly as both effector cells as well as cells with immunoregulatory potential.     

Isolation and culture of hepatocytes from human liver of immediate autopsy

Summary Human livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function. Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intack lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 × 10⁶ cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes, 632 pmol/mg protein of cytochromep ₄₅₀ and 536. pmol/mg protein cytochromeb ₅ were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture. This work was supported by EPA Grants R-809835-01-1, R-809599010 and DOE Contract DE-A505-83ER60158. Cobtribution no. 1762 from the Cellular Pathobiology Laboratory, University of Maryland School of Medicine.     

Drought‐resistant fungi control soil organic matter decomposition and its response to temperature

Microbial‐mediated decomposition of soil organic matter (SOM) ultimately makes a considerable contribution to soil respiration, which is typically the main source of CO₂ arising from terrestrial ecosystems. Despite this central role in the decomposition of SOM, few studies have been conducted on how climate change may affect the soil microbial community and, furthermore, on how possible climate‐change induced alterations in the ecology of microbial communities may affect soil CO₂ emissions. Here we present the results of a seasonal study on soil microbial community structure, SOM decomposition and its temperature sensitivity in two representative Mediterranean ecosystems where precipitation/throughfall exclusion has taken place during the last 10 years. Bacterial and fungal diversity was estimated using the terminal restriction fragment length polymorphism technique. Our results show that fungal diversity was less sensitive to seasonal changes in moisture, temperature and plant activity than bacterial diversity. On the other hand, fungal communities showed the ability to dynamically adapt throughout the seasons. Fungi also coped better with the 10 years of precipitation/throughfall exclusion compared with bacteria. The high resistance of fungal diversity to changes with respect to bacteria may open the controversy as to whether future ‘drier conditions’ for Mediterranean regions might favor fungal dominated microbial communities. Finally, our results indicate that the fungal community exerted a strong influence over the temporal and spatial variability of SOM decomposition and its sensitivity to temperature. The results, therefore, highlight the important role of fungi in the decomposition of terrestrial SOM, especially under the harsh environmental conditions of Mediterranean ecosystems, for which models predict even drier conditions in the future.