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1.  Spleen tyrosine kinase Syk is critical for sustained leukocyte adhesion during inflammation in vivo 
BMC Immunology  2007;8:31.
During inflammation, β2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk.
We investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk-/- hematopoietic system. In unstimulated venules, Syk-/- leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk-/- and control leukocytes. During fMLP-superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk-/- leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk-/- leukocytes. Additional in vitro experiments revealed that the adhesion and spreading defect seen in Syk-/- chimeric mice was due to compromised β2-integrin-mediated outside-in signaling.
We provide substantial evidence for an important role of Syk in mediating β2-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo.
PMCID: PMC2217554  PMID: 18045459
2.  CD43 signals induce Type One lineage commitment of human CD4+ T cells 
BMC Immunology  2007;8:30.
The activation and effector phenotype of T cells depend on the strength of the interaction of the TcR with its cognate antigen and additional signals provided by cytokines and by co-receptors. Lymphocytes sense both the presence of an antigen and also clues from antigen-presenting cells, which dictate the requisite response. CD43 is one of the most abundant molecules on the surface of T cells; it mediates its own signalling events and cooperates with those mediated by the T cell receptor in T cell priming. We have examined the role of CD43 signals on the effector phenotype of adult CD4+ and CD8+ human T cells, both alone and in the presence of signals from the TcR.
CD43 signals direct the expression of IFNγ in human T cells. In freshly isolated CD4+ T cells, CD43 signals potentiated expression of the IFNγ gene induced by TcR activation; this was not seen in CD8+ T cells. In effector cells, CD43 signals alone induced the expression of the IFNγ gene in CD4+ T cells and to a lesser extent in CD8+ cells. The combined signals from CD43 and the TcR increased the transcription of the T-bet gene in CD4+ T cells and inhibited the transcription of the GATA-3 gene in both populations of T cells, thus predisposing CD4+ T cells to commitment to the T1 lineage. In support of this, CD43 signals induced a transient membrane expression of the high-affinity chains of the receptors for IL-12 and IFNγ in CD4+ T cells. CD43 and TcR signals also cooperated with those of IL-12 in the induction of IFNγ expression. Moreover, CD43 signals induced the co-clustering of IFNγR and the TcR and cooperated with TcR and IL-12 signals, triggering a co-capping of both receptors in CD4+ populations, a phenomenon that has been associated with a T1 commitment.
Our results suggest a key role for CD43 signals in the differentiation of human CD4+ T cells into a T1 pattern.
PMCID: PMC2235884  PMID: 18036228
3.  Co-inherited mutations of Fas and caspase-10 in development of the autoimmune lymphoproliferative syndrome 
BMC Immunology  2007;8:28.
Autoimmune lymphoproliferative syndrome (ALPS) is a rare inherited disorder characterized by defective function of Fas, autoimmune manifestations that predominantly involve blood cells, polyclonal accumulation of lymphocytes in the spleen and lymph nodes with lymphoadenomegaly and/or splenomegaly, and expansion of TCRαβ+ CD4/CD8 double-negative (DN) T cells in the peripheral blood. Most frequently, it is due to Fas gene mutations, causing ALPS type Ia (ALPS-Ia). However, other mutations, namely of the FasL gene (ALPS-Ib) and the caspase-10 gene (ALPS-II) are occasionally detected, whereas some patients do not present any known mutations (ALPS-III). Recently, mutations of the NRAS gene have been suggested to cause ALPS-IV.
This work reports two patients that are combined heterozygous for single nucleotide substitutions in the Fas and caspase-10 genes. The first patient carried a splice site defect suppressing allele expression in the Fas gene and the P501L substitution in caspase-10. The second had a mutation causing a premature stop codon (Q47X) in the Fas gene and the Y446C substitution in caspase-10. Fas expression was reduced and caspase-10 activity was decreased in both patients. In both patients, the mutations were inherited from distinct healthy parents.
These data strongly suggest that co-transmission of these mutation was responsible for ALPS.
PMCID: PMC2211507  PMID: 17999750
4.  A unique population of effector memory lymphocytes identified by CD146 having a distinct immunophenotypic and genomic profile 
BMC Immunology  2007;8:29.
CD146 is a well described homotypic adhesion molecule found on endothelial cells and a limited number of other cell types. In cells from the peripheral circulation, CD146 has also been reported to be on activated lymphocytes in vitro and in vivo. The function associated with CD146 expression on lymphoid cells is unknown and very little information is available concerning the nature of CD146+ lymphocytes. In the current study, lymphocytes from healthy donors were characterized based upon the presence or absence of CD146 expression.
CD146 was expressed on a low percentage of circulating T lymphocytes, B lymphocytes, and NK cells in healthy individuals. CD146 expression can be induced and upregulated in vitro on both B cells and T cells, but does not correlate with the expression of other markers of T cell activation. CD146 positive T cells do not represent clonal expansions as determined with the use of anti Vβ reagents. Data suggest that CD146 positive cells have enhanced adherence to endothelial monolayers in vitro. Gene profiling and immunophenotyping studies between CD146+ and CD146- T cells revealed several striking genotypic distinctions such as the upregulation of IL-8 and phenotypic differences including the paucity of CCR7 and CD45RA among CD146 positive T cells, consistent with effector memory function. A number of genes involved in cell adhesion, signal transduction, and cell communication are dramatically upregulated in CD146+ T cells compared to CD146- T cells.
CD146 appears to identify small, unique populations of T as well as B lymphocytes in the circulation. The T cells have immunophenotypic characteristics of effector memory lymphocytes. The characteristics of these CD146+ lymphocytes in the circulation, together with the known functions in cell adhesion of CD146 on endothelial cells, suggests that these lymphocytes may represent a small subpopulation of cells primed to adhere to the endothelium and possibly extravasate to sites of inflammation.
PMCID: PMC2248207  PMID: 17999761
5.  A guide to modern statistical analysis of immunological data 
BMC Immunology  2007;8:27.
The number of subjects that can be recruited in immunological studies and the number of immunological parameters that can be measured has increased rapidly over the past decade and is likely to continue to expand. Large and complex immunological datasets can now be used to investigate complex scientific questions, but to make the most of the potential in such data and to get the right answers sophisticated statistical approaches are necessary. Such approaches are used in many other scientific disciplines, but immunological studies on the whole still use simple statistical techniques for data analysis.
The paper provides an overview of the range of statistical methods that can be used to answer different immunological study questions. We discuss specific aspects of immunological studies and give examples of typical scientific questions related to immunological data. We review classical bivariate and multivariate statistical techniques (factor analysis, cluster analysis, discriminant analysis) and more advanced methods aimed to explore causal relationships (path analysis/structural equation modelling) and illustrate their application to immunological data. We show the main features of each method, the type of study question they can answer, the type of data they can be applied to, the assumptions required for each method and the software that can be used.
This paper will help the immunologist to choose the correct statistical approach for a particular research question.
PMCID: PMC2234437  PMID: 17963513
6.  Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists 
BMC Immunology  2007;8:26.
Plasmacytoid Dendritic Cells (pDC) comprise approximately 0.2 to 0.8% of the blood mononuclear cells and are the primary type 1 interferon (IFN), producing cells, secreting high levels of IFN in response to viral infections. Plasmacytoid dendritic cells express predominantly TLRs 7 & 9, making them responsive to ssRNA and CpG DNA. The objective of this study was to evaluate the molecular and cellular processes altered upon stimulation of pDC with synthetic TLR 7 and TLR 7/8 agonists. To this end, we evaluated changes in global gene expression upon stimulation of 99.9% pure human pDC with the TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011.
Global gene expression was evaluated using the Affymetrix U133A GeneChip® and selected genes were confirmed using real time TaqMan® RTPCR. The gene expression profiles of the two agonists were similar indicating that changes in gene expression were solely due to stimulation through TLR7. Type 1 interferons were among the highest induced genes and included IFNB and multiple IFNα subtypes, IFNα2, α5, α6, α8, α1/13, α10, α14, α16, α17, α21. A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC. Induction of an antiviral state was shown by the expression of several IFN-inducible genes with known anti-viral activity. Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted. The analysis revealed transcription networks that show increased expression of signaling components in TLR7 and TLR3 pathways, and the cytosolic anti-viral pathway regulated by RIG1 and MDA5, suggestive of optimization of an antiviral state targeted towards RNA viruses. The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro-survival gene expression program.
Thus this study demonstrates that as early as 4 hr post stimulation, synthetic TLR7 agonists induce a complex transcription network responsible for activating pDC for innate anti-viral immune responses with optimized responses towards RNA viruses, increased co-stimulatory capacity, and increased survival.
PMCID: PMC2175514  PMID: 17935622
7.  Gene expression trees in lymphoid development 
BMC Immunology  2007;8:25.
The regulatory processes that govern cell proliferation and differentiation are central to developmental biology. Particularly well studied in this respect is the lymphoid system due to its importance for basic biology and for clinical applications. Gene expression measured in lymphoid cells in several distinguishable developmental stages helps in the elucidation of underlying molecular processes, which change gradually over time and lock cells in either the B cell, T cell or Natural Killer cell lineages. Large-scale analysis of these gene expression trees requires computational support for tasks ranging from visualization, querying, and finding clusters of similar genes, to answering detailed questions about the functional roles of individual genes.
We present the first statistical framework designed to analyze gene expression data as it is collected in the course of lymphoid development through clusters of co-expressed genes and additional heterogeneous data. We introduce dependence trees for continuous variates, which model the inherent dependencies during the differentiation process naturally as gene expression trees. Several trees are combined in a mixture model to allow inference of potentially overlapping clusters of co-expressed genes. Additionally, we predict microRNA targets.
Computational results for several data sets from the lymphoid system demonstrate the relevance of our framework. We recover well-known biological facts and identify promising novel regulatory elements of genes and their functional assignments. The implementation of our method (licensed under the GPL) is available at .
PMCID: PMC2244641  PMID: 17925013
8.  Mycobacterium tuberculosis 6-kDa Early Secreted Antigenic Target (ESAT-6) protein downregulates Lipopolysaccharide induced c-myc expression by modulating the Extracellular Signal Regulated Kinases 1/2 
BMC Immunology  2007;8:24.
Mycobacterium tuberculosis (Mtb) causes death of 2–3 million people every year. The persistence of the pathogenic mycobacteria inside the macrophage occurs through modulation of host cell signaling which allows them, unlike the other non-pathogenic species, to survive inside the host. The secretory proteins of M. tuberculosis have gained attention in recent years both as vaccine candidates and diagnostic tools; they target the immune system and trigger a putatively protective response; however, they may also be involved in the clinical symptoms of the disease.
Our studies showed that RD-1-encoded secretory protein ESAT-6 is involved in modulation of the mitogen-activated protein (MAP) kinase-signaling pathway inside the macrophage. ESAT-6 induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the cytoplasm but not in the nucleus, which normally is the case for MAP kinases. ESAT-6 also antagonized LPS-induced ERK1/2 phosphorylation in the nucleus. Stimulation of cells by ESAT-6 along with sodium orthovanadate (a tyrosine phosphatase inhibitor) restored phosphorylation of ERK1/2 in the nucleus, suggesting active dephosphorylation of ERK1/2 by some putative phosphatase(s) in the nucleus. Further, ESAT-6 was found to down regulate the expression of LPS-inducible gene c-myc in an ERK1/2-dependent manner.
This study showed the effect of secretory proteins of M. tuberculosis in the modulation of macrophage signaling pathways particularly ERK1/2 MAP kinase pathway. This modulation appears to be achieved by limiting the ERK1/2 activation in the nucleus which ultimately affects the macrophage gene expression. This could be a mechanism by which secretory proteins of Mtb might modulate gene expression inside the macrophages.
PMCID: PMC2082026  PMID: 17915024
9.  Production of monoclonal antibodies reactive with ovine eosinophils 
BMC Immunology  2007;8:23.
There is strong evidence implicating eosinophils in host defence against parasites as well as allergic disease pathologies. However, a lack of reagents such as monoclonal antibodies (mAbs) specific for eosinophils has made it difficult to confirm the functional role of eosinophils in such disease conditions. Using an established mammary model of allergic inflammation in sheep, large numbers of inflammatory cells enriched for eosinophils were collected from parasite-stimulated mammary glands and used for the generation of mAbs against ovine eosinophils.
A panel of mAbs was raised against ovine eosinophils of which two were shown to be highly specific for eosinophils. The reactivity of mAbs 3.252 and 1.2 identified eosinophils from various cell and tissue preparations with no detectable reactivity on cells of myeloid or lymphoid lineage, tissue mast cells, dendritic cells, epithelial cells or other connective tissues. Two other mAbs generated in this study (mAbs 4.4 and 4.10) were found to have reactivity for both eosinophils and neutrophils.
This study describes the production of new reagents to identify eosinophils (as well as granulocytes) in sheep that will be useful in studying the role of eosinophils in disease pathologies in parasite and allergy models.
PMCID: PMC2045663  PMID: 17897475
10.  Haematopoietic development and immunological function in the absence of cathepsin D 
BMC Immunology  2007;8:22.
Cathepsin D is a well-characterized aspartic protease expressed ubiquitously in lysosomes. Cathepsin D deficiency is associated with a spectrum of pathologies leading ultimately to death. Cathepsin D is expressed at high levels in many cells of the immune system, but its role in immune function is not well understood. This study examines the reconstitution and function of the immune system in the absence of cathepsin D, using bone marrow radiation chimaeras in which all haematopoietic cells are derived from cathepsin D deficient mice.
Cathepsin D deficient bone marrow cells fully reconstitute the major cellular components of both the adaptive and innate immune systems. Spleen cells from cathepsin D deficient chimaeric mice contained an increased number of autofluorescent granules characteristic of lipofuscin positive lysosomal storage diseases. Biochemical and ultrastructural changes in cathepsin D deficient spleen are consistent with increased autolysosomal activity. Chimaeric mice were immunised with either soluble (dinitrophenylated bovine gamma globulin) or particulate (sheep red blood cells) antigens. Both antigens induced equivalent immune responses in wild type or cathepsin D deficient chimaeras.
All the parameters of haematopoietic reconstitution and adaptive immunity which were measured in this study were found to be normal in the absence of cathepsin D, even though cathepsin D deficiency leads to dysregulation of lysosomal function.
PMCID: PMC2048983  PMID: 17897442
11.  Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses 
BMC Immunology  2007;8:21.
Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response.
Linear regression analysis indicates that both stimulation index (SI) (p = 0.011) and IFN-gamma secreting precursor frequency (p < 0.001) are significant indicators of antigen specific immunity. ROC curves plotted to assess the performance of tritiated thymidine incorporation and the ELISPOT assay indicate that SI is a significant indicator of low T cell response to the HER-2/neu vaccine (p = 0.05), and of moderate and robust responses to tt (p = 0.01) and CMV (p = 0.016), respectively. IFN-gamma precursor frequency is a significant indicator of a robust T cell response to CMV (p = 0.03), but not of moderate tt (p = 0.09), or low HER-2/neu (p = 0.09) T cell responses.
These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.
PMCID: PMC2034595  PMID: 17850666
12.  Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis 
BMC Immunology  2007;8:20.
The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene™ Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use.
The aim of this study was to modify the PAXgene™ Blood RNA System kit protocol for application to small, sick chidren, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression.
Aliquots of 0.86 mL PAXgene™ reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene™ evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan™ assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease.
Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02).
We have successfully modified the PAXgene™ blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis.
PMCID: PMC2031894  PMID: 17850649
13.  Milk fermentation products of L. helveticus R389 activate calcineurin as a signal to promote gut mucosal immunity 
BMC Immunology  2007;8:19.
Fermented milks containing probiotic bacteria are a way of delivering bioactive constituents to targets in the gastrointestinal tract. We reported previously that the fermentation of milk at constant pH 6 by L. helveticus R389 increased its content of peptide fractions, and the oral administration of the non-bacterial fraction (FMSpH6) to mice increased total secretory IgA in the intestinal lumen and enhanced the number of IgA and various cytokines producing cells as well as the secretion of IL-6 by small intestine epithelial cells. We also demonstrated that this FMSpH6 was effective for the prevention of Salmonella typhimurium infection in mice. In this work, we studied in mice the impact of the oral administration of the supernatant of milk fermented by L. helveticus R389 on the gut physiology by measuring parameters such as calcium channels and E-cadherin expression, the activation of the biological signal calcineurin and mast and goblet cells, as a way to determine some mechanisms involved in the immunomodulating effects of the milk fermentation products, observed in previous studies. We analyzed the impact of the supernatant of milk fermented by L. helveticus R389 at pH6-controlled on the expression of calcineurin and on the reinforcement of the ephitelial barrier, measuring parameters such as calcium channels and E-cadherin expression and in the reinforcement of the non-specific immunity determining mast cells and goblet cells associated to the gut.
We observed an enhanced expression of TRPV6 channels in the duodenum, indicating an improved capacity for dietary Ca2+ uptake. We demonstrated an enhanced expression of calcineurin in the small intestine, able to upregulate immune parameters such as IL-2 and TNF production, with an increase in the number of these cytokines secreting cells. We determined an increase in the number of mucosal mast cells and goblet cells, which would mean an improved state of mucosal surveillance at sites of infection.
The oral administration of the supernatant of milk fermented by L. helveticus R389 enhanced the gut mucosal immunity by improving the mechanisms that reinforce the epithelial and non-specific barriers and the gut functioning at sites of infection, with an improvement in the expression of the enzyme calcineurin, an important signal in the network that activates the gut immune system. The results of this work contribute to revealing the mechanisms underlying the immunomodulation of the gut immune function by fermented milks with probiotic bacteria.
PMCID: PMC2045662  PMID: 17825099
14.  Cytokine profiles of cord and adult blood leukocytes: differences in expression are due to differences in expression and activation of transcription factors 
BMC Immunology  2007;8:18.
Stem cell transplantation as therapy for hematological disorders is often hampered by severe graft-versus-host-disease. This may be reduced by umbilical cord blood transplantation, an effect that has been attributed to qualitative differences between neonatal and adult T cells. We compared levels of secreted proteins and cytokine mRNA induced in cord blood leukocytes (CBL) and adult blood leukocytes (ABL) by various stimuli.
While interleukin-2 (IL-2) levels were similar in CBL and ABL, there was less induction of the Th1 cytokine interferon-γ in CBL. Production of the Th2 cytokines IL-4, IL-5, and IL-13 and the hematopoietic cytokine IL-3 was much lower in CBL versus ABL after T-cell receptor-mediated stimulation, whereas production of GM-CSF was comparable in the 2 cell types. The lower levels of Th1 and Th2 cytokines were maintained in CBL during a 4-day time-course study, while after 12 hours IL-3 and GM-CSF reached in CBL levels similar to those in ABL. For all cytokines except IFNγ, the IC50 values for inhibition by cyclosporin A were similar in ABL and CBL. In contrast, there was less expression and activation of transcription factors in CBL. Activation of NF-κB by TPA/ionomycin was detected in ABL but not CBL. Furthermore, there was less expression of the Th subset-specific transcription factors T-bet and c-maf in CBL versus ABL, whereas GATA-3 expression was similar. Expression of T-bet and c-maf correlated with expression of the Th1 and Th2 cytokines, respectively. Time course experiments revealed that T-bet expression was stimulated in both cell types, whereas c-maf and GATA-3 were induced only in ABL.
The diminished capability of CBL to synthesize cytokines is probably due to decreased activation of NF-κB, whereas differences in Th subsets are due to differences in regulation of Th lineage-specific transcriptions factors. We propose that the reduced incidence and severity of GvHD after allogeneic transplantation of umbilical CB cells is due to lesser activation of specific transcription factors and a subsequent reduction in production of certain cytokines.
PMCID: PMC2018703  PMID: 17764543
15.  Transcription factor network downstream of protease activated receptors (PARs) modulating mouse bladder inflammation 
BMC Immunology  2007;8:17.
All four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders.
For this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kitw/Kitw-v) mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB.
TFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, Kitw/Kitw-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kitw/Kitw-v mice.
This is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders.
PMCID: PMC2000913  PMID: 17705868
16.  Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells 
BMC Immunology  2007;8:14.
Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an essential role in the host defence against intracellular pathogens such as Listeria, and Mycobacteria. The key mediator of bacteria-directed cytotoxicity is granulysin, a 9 kDa protein stored in cytolytic granules together with perforin and granzymes. Granulysin binds to cell membranes and is subsequently taken up via a lipid raft-associated mechanism. In dendritic cells (DC) granulysin is further transferred via early endosomes to L. innocua-containing phagosomes were bacteriolysis is induced. In the present study we analysed the role of perforin in granulysin-induced intracellular bacteriolysis in DC.
We found granulysin-induced lysis of intracellular Listeria significantly increased when perforin was simultaneously present. In pulse-chase experiments enhanced bacteriolysis was observed when perforin was added up to 25 minutes after loading the cells with granulysin demonstrating no ultimate need for simultaneous uptake of granulysin and perforin. The perforin concentration sufficient to enhance granulysin-induced intracellular bacteriolysis did not cause permanent membrane pores in Listeria-challenged DC as shown by dye exclusion test and LDH release. This was in contrast to non challenged DC that were more susceptible to perforin lysis. For Listeria-challenged DC, there was clear evidence for an Ca2+ influx in response to sublytic perforin demonstrating a short-lived change in the plasma membrane permeability. Perforin treatment did not affect granulysin binding, initial uptake or intracellular trafficking to early endosomes. However, enhanced colocalization of granulysin with listerial DNA in presence of perforin was found by confocal laser scanning microscopy.
The results provide evidence that perforin increases granulysin-mediated killing of intracellular Listeria by enhanced phagosome-endosome fusion triggered by a transient Ca2+ flux.
PMCID: PMC1976101  PMID: 17705829
17.  Direct cell-to-cell spread of a pathogenic yeast 
BMC Immunology  2007;8:15.
Cryptococcosis, a fatal fungal infection of the central nervous system, is one of the major killers of AIDS patients and other immunocompromised hosts. The causative agent, Cryptococcus neoformans, has a remarkable ability to 'hide' and proliferate within phagocytic cells of the human immune system. This intracellular phase is thought to underlie the ability of the pathogen to remain latent for long periods of time within infected individuals.
We now report that Cryptococcus is able to undergo 'lateral transfer' between phagocytes, moving directly from infected to uninfected macrophages. This novel process was observed in both C. neoformans serotypes (A and D) and occurs in both immortalised cell lines and in primary human macrophages. Lateral transfer is independent of the initial route of uptake, since both serum-opsonised and antibody-opsonised C. neoformans are able to undergo direct cell-to-cell transfer.
We provide the first evidence for lateral transfer of a human fungal pathogen. This rare event may occur repeatedly during latent cryptococcal infections, thereby allowing the pathogen to remain concealed from the immune system and protecting it from exposure to antifungal agents.
PMCID: PMC1976318  PMID: 17705831
18.  Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages 
BMC Immunology  2007;8:16.
The interaction between macrophages and Cryptococcus neoformans (Cn) is critical for containing dissemination of this pathogenic yeast. However, Cn can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. Both events result in live extracellular yeasts capable of reproducing and disseminating in the extracellular milieu. Another method of exiting the intracellular confines of cells is through host cell-to-cell transfer of the pathogen, and this commonly occurs with the human immuno-deficiency virus (HIV) and CD4+ T cells and macrophages. In this report we have used time-lapse imaging to determine if this occurs with Cn.
Live imaging of Cryptococcus neoformans interactions with murine macrophages revealed cell-to-cell spread of yeast cells from infected donor cells to uninfected cells. Although this phenomenon was relatively rare its occurrence documents a new capacity for this pathogen to infect adjacent cells without exiting the intracellular space. Cell-to-cell spread appeared to be an actin-dependent process. In addition, we noted that cryptococcal phagosomal extrusion was followed by the formation of massive vacuoles suggesting that intracellular residence is accompanied by long lasting damage to host cells.
C. neoformans can escape the intracellular confines of macrophages in an actin dependent manner by cell-to-cell transfer of the yeast leading to infection of adjacent cells. In addition, complete extrusion of internalized Cn cells can lead to the formation of a massive vacuole which may be a sign of damage to the host macrophage. These observations document new outcomes for the interaction of C. neoformans with host cells that provide precedents for cell biological effects that may contribute to the pathogenesis of cryptococcal infections.
PMCID: PMC1988836  PMID: 17705844
19.  Impact of polysialylated CD56 on natural killer cell cytotoxicity 
BMC Immunology  2007;8:13.
Siglec-7, a sialic acid binding inhibitory receptor expressed by NK cells is masked in vivo by a so far unknown ligand. It shows a strong binding prevalence for α-2,8-linked disialic acids in vitro.
Here we describe the expression of PSA-NCAM (α-2,8-linked polysialic acid modified NCAM) on functional adult peripheral blood natural killer cells and examine its possible role in masking Siglec-7. Unmasking of Siglec-7 using Clostridium perfringens neuraminidase massively reduces NK cell cytotoxicity. By contrast a specific removal of PSA using Endo-NF does not lead to a reduction of NK cell cytotoxicity.
The results presented here therefore indicate that PSA-NCAM is not involved in masking Siglec-7.
PMCID: PMC1976417  PMID: 17683591
20.  CD8α is expressed by human monocytes and enhances FcγR-dependent responses 
BMC Immunology  2007;8:12.
CD8α enhances the responses of antigen-specific CTL activated through TCR through binding MHC class I, favoring lipid raft partitioning of TCR, and inducing intracellular signaling. CD8α is also found on dendritic cells and rat macrophages, but whether CD8α enhances responses of a partner receptor, like TCR, to activate these cells is not known. TCR and FcR, use analogous or occasionally interchangeable signaling mechanisms suggesting the possibility that CD8α co-activates FcR responses. Interestingly, CD8α+ monocytes are often associated with rat models of disease involving immune-complex deposition and FcR-mediated pathology, such as arthritis, glomerulonephritis, ischaemia, and tumors. While rat macrophages have been shown to express CD8α evidence for CD8α expression by mouse or human monocytes or macrophages was incomplete.
We detected CD8α, but not CD8β on human monocytes and the monocytic cell line THP-1 by flow cytometry. Reactivity of anti-CD8α mAb with monocytes is at least partly independent of FcR as anti-CD8α mAb detect CD8α by western blot and inhibit binding of MHC class I tetramers. CD8α mRNA is also found in monocytes and THP-1 suggesting CD8α is synthesized by monocytes and not acquired from other CD8α+ cell types. Interestingly, CD8α from monocytes and blood T cells presented distinguishable patterns by 2-D electrophoresis. Anti-CD8α mAb alone did not activate monocyte TNF release. In comparison, TNF release by human monocytes stimulated in a FcR-dependent manner with immune-complexes was enhanced by inclusion of anti-CD8α mAb in immune-complexes.
Human monocytes express CD8α. Co-engagement of CD8α and FcR enhances monocyte TNF release, suggesting FcR may be a novel partner receptor for CD8α on innate immune cells.
PMCID: PMC2000912  PMID: 17678538
21.  TSLP is involved in expansion of early thymocyte progenitors 
BMC Immunology  2007;8:11.
Thymic stromal derived lymphopoietin (TSLP) is preferentially and highly expressed in the thymus, but its function in T cell development is not clear.
We report here that TSLP, independently or in combination with IL-7, enhances thymopoiesis in the murine fetal thymic organ culture (FTOC) model. Furthermore, TSLP preferentially increases the number and proliferation of the (DN1 and DN2) pro-T progenitor cells, and FTOC lobes from TSLP receptor-null mice show a decreased number of these cells. Finally, DN1-DN2 cells expanded with TSLP in vitro are functional T progenitors that are able to differentiate into mature T cells in fetal or adult thymus organs.
Together, these data suggest that TSLP plays an important role in expansion of thymocyte progenitors and may be of value for expanding T progenitor cells in vitro.
PMCID: PMC1940266  PMID: 17640367
22.  Modulation of microglial/macrophage activation by macrophage inhibitory factor (TKP) or tuftsin (TKPR) attenuates the disease course of experimental autoimmune encephalomyelitis 
BMC Immunology  2007;8:10.
Myelin Oligodendrocyte Glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used mouse model for multiple sclerosis (MS). During the of progression of EAE, microglia, the immunocompetent cells of the brain, become activated and accumulate around demyelinated lesions. Microglial activation is mediated by the extracellular protease tissue Plasminogen Activator (tPA), and mice lacking tPA display altered EAE progression. In this study, we have used pharmacological inhibitors and stimulators of microglial/macrophage activation to examine the temporal requirement for microglial activation in EAE progression and to determine whether such approaches might potentially be of therapeutic value.
Intervention using the tripeptide macrophage/microglia inhibitory factor MIF (TKP) and the tetrapeptide macrophage/microglial stimulator tuftsin (TKPR) attenuated EAE symptoms and revealed that the timing of macrophage/microglial activation is critical for the clinical outcome of EAE. We show that the disease progression can potentially be manipulated favorably at early stages by altering the timing of microglial activation, which in turn alters the systemic immune response to favor upregulation of T helper cell 2 genes that promote recovery from EAE.
Preventative and therapeutic modulation of macrophage/microglial activity significantly alters the outcome of EAE at symptomatic stages. Specific molecular targets have been identified that represent potential avenues of exploration for the treatment and prevention of MS.
PMCID: PMC1937009  PMID: 17634104
23.  Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes 
BMC Immunology  2007;8:9.
Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein.
In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation.
Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells.
This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing.
PMCID: PMC1914365  PMID: 17596262
24.  Interleukin-10 plays an early role in generating virus-specific T cell anergy 
BMC Immunology  2007;8:8.
Infection of mice with the Armstrong strain of lymphocytic choriomeningitis virus (LCMVARM) leads to a robust immune response and efficient viral clearance. This is in contrast to infection with the variant strain LCMVClone13, which causes functional inactivation of effector T cells and viral persistence. The mechanism by which LCMVClone13 suppresses the antiviral immune response and persists in its host is unknown.
Here we demonstrate that infection with LCMVClone13, but not with LCMVARM, resulted in a steady increase in the serum levels of the immuno-inhibitory cytokine, IL-10. Blockade of IL-10 using neutralizing monoclonal antibody injections in LCMVClone13-infected mice led to dramatically enhanced effector T cell responses at 8 days post-infection. Even though IL-10 blockade resulted in decreased viral titers, the generation and maintenance of memory T cells was still compromised. The functional inactivation of CD8+ T cells in IL-10-blocked, chronically infected mice 30 days post-infection was incomplete as potent CTL (cytotoxic T lymphocytes) could be generated by in vitro re-stimulation. IL-10 knockout mice showed a similar pattern of antiviral CD8 T cell responses: early antiviral T cells were dramatically increased and viral levels were decreased; however, CD8 T cells in IL-10 knockout mice were also eventually anergized and these mice became persistently infected.
Our data suggest that IL-10 plays an early role in LCMVClone13-induced tolerance, although other factors collaborate with IL-10 to induce virus-specific tolerance.
PMCID: PMC1903364  PMID: 17570849
25.  Immune system changes during simulated planetary exploration on Devon Island, high arctic 
BMC Immunology  2007;8:7.
Dysregulation of the immune system has been shown to occur during spaceflight, although the detailed nature of the phenomenon and the clinical risks for exploration class missions have yet to be established. Also, the growing clinical significance of immune system evaluation combined with epidemic infectious disease rates in third world countries provides a strong rationale for the development of field-compatible clinical immunology techniques and equipment. In July 2002 NASA performed a comprehensive immune assessment on field team members participating in the Haughton-Mars Project (HMP) on Devon Island in the high Canadian Arctic. The purpose of the study was to evaluate the effect of mission-associated stressors on the human immune system. To perform the study, the development of techniques for processing immune samples in remote field locations was required. Ten HMP-2002 participants volunteered for the study. A field protocol was developed at NASA-JSC for performing sample collection, blood staining/processing for immunophenotype analysis, whole-blood mitogenic culture for functional assessments and cell-sample preservation on-location at Devon Island. Specific assays included peripheral leukocyte distribution; constitutively activated T cells, intracellular cytokine profiles, plasma cortisol and EBV viral antibody levels. Study timepoints were 30 days prior to mission start, mid-mission and 60 days after mission completion.
The protocol developed for immune sample processing in remote field locations functioned properly. Samples were processed on Devon Island, and stabilized for subsequent analysis at the Johnson Space Center in Houston. The data indicated that some phenotype, immune function and stress hormone changes occurred in the HMP field participants that were largely distinct from pre-mission baseline and post-mission recovery data. These immune changes appear similar to those observed in astronauts following spaceflight.
The immune system changes described during the HMP field deployment validate the use of the HMP as a ground-based spaceflight/planetary exploration analog for some aspects of human physiology. The sample processing protocol developed for this study may have applications for immune studies in remote terrestrial field locations. Elements of this protocol could possibly be adapted for future in-flight immunology studies conducted during space missions.
PMCID: PMC1890299  PMID: 17521440

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