Cerebral hypoxia-ischemia and middle cerebral artery occlusion induce expression of the cannabinoid CB2 receptor in the brain

Until recently the cannabinoid CB2 receptor was believed to be absent from the central nervous system. In this study we have identified CB2 expressing cells that appear in the rat brain following stroke and hypoxic-ischemia. At 3 days following surgery CB2-positive macrophages, deriving from resident microglia and/or invading monocytes appear on the lesioned side of the brain. By day 7, a mixed population of CB2-positive cells is present. Microglia-derived macrophages are the key cells in the first stages of brain inflammation, and a pivotal step in the neurodegeneration that follows the acute stage of injury. Thus, CB2 may be important in the brain during injury, and in inflammatory neurodegenerative disorders. The presence of CB2-positive cells in the brain following stroke may provide a novel strategy for cannabinoid-mediated intervention into stroke induced neurodegeneration without the psychoactive effects of CB1 receptor stimulation.     

The human beta-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal beta-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the beta-glucan receptor (betaGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional betaGR isoforms - betaGR-A and betaGR-B - were expressed by these cell populations in peripheral blood; however, only betaGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other beta-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major beta-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.     

The TRAF6, but not the TRAF2/3, binding domain of CD40 is required for cytokine production in human lung fibroblasts

Fibroblasts are key effector cells in inciting inflammation, wound healing, and scarring. CD40, a member of the TNF receptor superfamily, mediates intercellular communication between fibroblasts and cells that express CD154 (CD40L), including T lymphocytes and platelets. To better understand the mechanisms by which CD40 regulates fibroblast function in inflammation and scarring, we examined the ability of CD40 cytoplasmic tail regions (CD40ct) containing the TRAF6 or the TRAF2/3 binding domains to regulate cytokine and chemokine expression by primary human lung fibroblasts. The full-length human CD40ct, the first 35 amino acids of the CD40ct encompassing the TRAF6 binding site (1-35), and amino acids 35-53 containing the TRAF2/TRAF3 binding domain were expressed in human lung fibroblasts as fusion proteins with the extracellular domain of human CD8alpha by retroviral transduction. The TRAF6, but not the TRAF2/3, binding domain was found to regulate IL-8 and IL-6 production, and induce activation of NF-kappaB and Jun kinase in lung fibroblasts, demonstrating for the first time that CD40ct domains can function independently to regulate pro-inflammatory responses of primary human fibroblasts. Thus, targeting TRAF6 function through pharmacological intervention may represent a viable strategy for modulating localized inflammation.     

Mast cell-expressed complement receptor, not TLR2, is the main detector of zymosan in peritonitis

The in vitro macrophage response to zymosan has been attributed to Toll-like receptor 2 (TLR2). Whether TLR2 is obligatory for the zymosan-induced in vivo response has not been assessed. The importance of this question is underscored by the fact that zymosan activates complement in a cell-independent manner. We have investigated whether the in vitro observation of TLR2 as the dominant zymosan receptor on macrophages would translate to an experimental peritonitis model in vivo. We have treated mice with zymosan, resulting in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. Zymosan-mediated leukocyte recruitment was TLR2 independent, but was predominantly dependent on the complement components, C3 and C5a with a minor contribution from LTB4. Peritoneal neutrophilia was 50% mast cell dependent and this defect was reproduced using C5a receptor (C5aR)-deficient mast cells in mast cell-deficient mice, suggesting that C5aR is responsible for mast cell activation following zymosan challenge. By 24 h, the response to zymosan involved primarily monocyte recruitment and was C3 and C5aR independent. Taken together, these studies indicate that the in vivo inflammatory response to zymosan does not necessarily mimic the TLR2 dependence observed in vitro, and that complement plays a dominant role in early, but not late, zymosan-mediated peritonitis.     

LAM-1/Leu 8 antigen is expressed on portal, but not on lobular intrahepatic mononuclear cells in inflammatory liver disease.

The expression of the selectin receptor LAM-1/Leu 8 was analysed in normal and in inflamed liver tissue, and its expression on mononuclear inflammatory cells was correlated with their topographical distribution in various compartments of the inflamed liver, in order to obtain new insights on possible molecular mechanisms involved in the traffic of mononuclear inflammatory cells throughout the diseased hepatic parenchyma. In normal liver tissue, few scattered mononuclear cells in portal and lobular parenchyma corresponded to both CD4+ and CD8+, as well as to CD45RA+ (2H4+) naive and CD45RO+ (UCHL1+) memory T cells, and were LAM-1/Leu 8+. In acute and chronic inflamed liver biopsies, CD45RO+ (UCHL1+) CD4+ and CD8+ memory T cells largely predominated in both portal and lobular parenchyma. The expression of LAM-1/Leu 8 antigen on these memory T cells varied according to their localization in the liver parenchyma, and it was not correlated with specific aetiological causes. In acute hepatitis, the vast majority of T lymphocytes were LAM-1/Leu 8-. In chronic active hepatitis, memory T cells in portal tracts expressed LAM-1/Leu 8, whereas virtually all intralobular T cells accumulating in areas of periportal and intralobular inflammation were LAM-1/Leu 8-. In chronic persistent hepatitis, the LAM-1/Leu 8+ T cells largely predominated among the numerous mononuclear inflammatory cells within enlarged portal tracts, whereas LAM-1/Leu 8- T cells were restricted to areas of intralobular 'spotty' inflammation. Therefore, two phenotypical populations can be recognized among the memory T cells in inflamed liver tissue, according to their topographical localization: LAM-1/Leu 8+ T cells predominating in portal tracts, and LAM-1/Leu 8- T cells predominating in the lobular parenchyma. These data show that during their migration through the inflamed liver parenchyma, memory T lymphocytes undergo phenotypical changes (LAM-1/Leu 8 shedding) according to their localization in different liver compartments (portal tracts vs. lobular parenchyma), suggesting multiple cellular and molecular mechanisms involved in the regulation of the leucocyte traffic through inflamed liver tissue.     

Only VpreB1, but not VpreB2, is expressed at levels which allow normal development of B cells

The surrogate light chain (SLC) consists of the polypeptides lambda5 and, in the mouse, either VpreB1 or VpreB2. SLC associates with BILL-Cadherin and other glycoproteins to form the pro-B cell receptor (pro-BCR) at the pre-BI cell stage, and with the immunoglobulin mu heavy chain to form the pre-BCR at the pre-BII cell stage. The function of the pro-BCR, if any, is unknown, whereas the pre-BCR is crucial for proliferative expansion of pre-BII cells. To shed light on the functional properties of VpreB1 and VpreB2 in vivo, mice with either one or two VpreB1, or one or two VpreB2, alleles have been investigated. We show that B cell development in mice with two VpreB1 alleles is indistinguishable from that of normal mice. In contrast, mice with two VpreB2 alleles show an approximately 1.6-fold increase in pre-BI and a 35% decrease in pre-BII cell numbers, while mice with only one VpreB2 allele show a reduction in B cell development manifested in a 2-fold enrichment in pre-BI cells and a 75% reduction in pre-BII cells. However, such a gene dosage effect is not observed for VpreB1. Our results suggest that the difference between VpreB1- and VpreB2-deficient mice is due to lower VpreB2 protein expression, thus limiting the formation of pre-BCRs and thereby the number of large, cycling pre-BII cells.     

MDR1 P-glycoprotein is expressed by endothelial cells of newly formed capillaries in human gliomas but is not expressed in the neovasculature of other primary tumors.

The expression of human MDR1 P-glycoprotein (Pgp) in the capillary endothelial cells of the central nervous system has been demonstrated. The brain capillary endothelial cells maintain the structure and function of the blood-brain barrier. Recently, the human MDR1 Pgp (and its mouse homologue MDR1a Pgp) has been shown to function as an important part of this barrier, pumping out xenobiotics from endothelial cells into the lumen of capillaries resulting in the protection of the brain parenchyma. To examine whether the endothelial cells of the newly formed capillaries during neoangiogenesis within malignant human brain tumors express MDR1 Pgp, 35 adult surgical brain tumor specimens (29 gliomas and 6 tumors metastatic to the brain) were obtained from previously untreated patients and studied by a new immunohistochemical sandwich method developed in our laboratory using the JSB-1 monoclonal antibody. JSB-1 is specific for the Pgp product of the human MDR1 (and not MDR3) gene. This sensitive method allows the detection of Pgp in capillary endothelial cells of normal brain in conventional paraffin sections after formalin fixation. The endothelial cells of the newly formed capillaries in 25 of 29 gliomas (86%) and 3 of 6 metastatic tumors, immunostained positive for MDR1 Pgp. The tumor cells in 7 of 35 cases were also positive for Pgp. In the 35 brain tumor cases investigated, the endothelial cells were Pgp positive in the tumor-brain border and in the brain further from the tumor. Capillary endothelial cells of neovasculature in 137 malignant tumors (non-brain) obtained from previously untreated patients showed no MDR1 Pgp expression. These results demonstrated that MDR1 Pgp is expressed not only in the capillaries of normal brain but also in the majority of the newly formed capillaries of brain tumors. Multidrug resistance of brain tumors may result not only from the expression of resistance markers in neoplastic cells but also from the MDR1 Pgp expression in endothelial cells of tumor capillaries. Pgp in this special localization can exclude chemotherapeutic agents from tumor cells that are located around the capillaries. The therapeutic benefit and selectivity of chemotherapeutic agents in combination with a Pgp-reversing agent should be evaluated.     

Interleukin-6, but not interleukin-4, is expressed by Reed-Sternberg cells in Hodgkin''s disease with or without histologic features of Castleman''s disease.

Hodgkin's disease (HD) is a neoplastic disease that is characterized by unbalanced and/or unregulated cytokine production. Information accumulated in our own and other laboratories indicates that the cytokines interleukin-1 (IL-1), IL-5, IL-9, tumor necrosis factor-alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), macrophage CSF (M-CSF), and transforming growth factor-beta (TGF-beta) are secreted by Hodgkin's and Reed-Sternberg (H-RS) cells. These and perhaps additional cytokines are likely to be responsible for the unique histopathologic and clinical alterations seen in patients with HD. In this study, we confirmed that IL-6 is produced by cultured H-RS cells as well as by H-RS cells in tissues. By using an enzyme-linked immunosorbent assay, we found that approximately 2 to 10 ng/ml of IL-6 was secreted by cultured H-RS cells (10(6) cells/ml). In tissues, we were able to immunolocalize IL-6 in the cytoplasm in 10 to 30% of H-RS cells by using rabbit polyclonal and mouse monoclonal anti-IL-6 antibodies. There was no correlation among the IL-6 staining intensity, number of H-RS cells stained, and the degree of plasma cell infiltration. However, in 3 of 17 cases studied, a large number (60%) of H-RS cells were positive for IL-6, and in these patients, abundant plasma cells were present. In one patient, the involved lymph node also showed histologic features similar to those of Castleman's disease. In this patient, we noted abundant IL-6 expression not only in H-RS cells, but also in most reactive histiocytes. The cultured H-RS cells did not express functional receptors for IL-6, and exogenously added IL-6 did not induce proliferation of these cells. We also conducted studies with specific anti-IL-4 antibodies, which did not show IL-4 production by H-RS cells in both cultures and tissues. In tissues, only rare IL-4 positive lymphoid cells or dendritic cells were identified. Thus, the study demonstrated that adequate amounts of IL-6 are required for an abundant plasma cell reaction, and that an additional source of IL-6 from histiocytes is essential for the formation of Castleman's disease-like changes in lymph nodes involved by HD. Furthermore, IL-4 is not likely to be responsible for the T-lymphocyte reaction in tissues, by a mechanism distinct from that in T-cell-rich B-cell lymphomas.     

The rat orthologue to the inhibitory receptor gp49B is expressed by neutrophils and monocytes, but not by NK cells or mast cells

Mouse gp49B is a member of the leukocyte immunoglobulin-like receptor family. It is constitutively expressed by mast cells and certain myeloid cells, and expression can be induced on natural killer (NK) cells and T cells. We have cloned several rat cDNA, 78% identical to mouse gp49B at the amino acid level, that represent the rat orthologue to mouse gp49B. A mouse monoclonal antibody (WEN29) against rat gp49B was generated. By flow cytometry and Northern blot analysis, gp49B was found to be expressed by neutrophils and monocytes, but not NK cells (primary or IL-2-activated), T cells (resting or concanavalin A-stimulated) or peritoneal mast cells. Following pervanadate treatment, the tyrosine phosphatase SHP-1 was co-immunoprecipitated with gp49B in the macrophage cell line R2. In glutathione S-transferase pull-down experiments, the cytoplasmic tail of rat gp49B associated with the SH2 domains of both SHP-1 and SHP-2, dependent on intact and phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIM). Compared to mouse, the cytoplasmic domain of rat gp49B contains a third ITIM-like sequence (YLYASV) that was phosphorylated by several Src family tyrosine kinases, enhanced the phosphorylation of other ITIM, and bound to the SH2 domains of SHP-2, suggesting a role in the recruitment of downstream phosphatases.     

Triggering receptor expressed on myeloid cells 2 variant is rare in late-onset Alzheimer's disease in Han Chinese individuals

Recent studies have reported that a rare mutation of triggering receptor expressed on myeloid cells 2 gene (TREM2 [rs75932628-T]) has significantly increased the risk of late-onset Alzhemier's disease (LOAD) in European-descendent population. To date, no study has investigated the association between rare mutations of TREM2 and LOAD risk in non-European population. Here, we sequenced exon2 of TREM2 in the northern Han Chinese population consisting of 1133 patients with LOAD and 1159 control subjects. Although, 4 novel mutations (c.102G>A: Val34Val, c.330C>T: Cys110Cys, c.342T>C: His114His, and c.343G>A: Gly115Ser) were identified in patients with LOAD, none of them exhibited significant association with LOAD risk after Bonferroni correction. Most importantly, the previously reported rare variants in European-descendent population including rs75932628-T (predicted to cause an R47H substitution) were absent in our cohort. These findings suggest that mutations in exon2 of TREM2 were unlikely to play a key role in the susceptibility of LOAD in the northern Han Chinese population.     

SOX-2, but not Oct4, is highly expressed in early-stage endometrial adenocarcinoma and is related to tumour grading

Background: Expression of SOX-2 and Oct4 as markers for the identification of cancer stem cells (CSCs) has been revealed in several malignancies. In this study, the co-expression of SOX-2 and Oct4 and their correlation with clinicopathological features of endometrial adenocarcinomas (EACs) was investigated. Methods: SOX-2 and Oct4 expression was assessed by immunohistochemistry in 27 (39.13%) stage IA and in 42 (60.87%) stage IB International Federation of Gynaecology and Obstetrics (FIGO) EACs and related to the clinicopathological features of patients. Results: The expression of SOX-2 was confirmed in 62/69 tumour specimens compared to Oct4 expression in 46/69 specimens (P = 0.015) and no difference in median staining intensity between SOX-2 and Oct-4 was observed. The highest median SOX-2 expression was found in high-grade (G3) EAC samples compared to moderate-grade (G2) EAC specimens (P = 0.020) and low-grade (G1) specimens (P = 0.008), while no differences in median Oct4 expression in EAC samples according to grading were present. In G3 specimens, significantly higher median SOX-2 expression was noted compared to Oct4 (P = 0.002). SOX-2 and Oct4 co-expression was observed only in G1 EAC (R: 0.51; P = 0.031). Age of EAC diagnosis was positively correlated with SOX-2 expression (b = 0.193; R² = 10.83%; P = 0.003) but not to age of menarche, menopause, parity or body mass index. Conclusions: There is no need to use SOX-2 expression as a poor outcome predictor in stage I EAC, and SOX-2 expression should be analysed in more advanced stages.     

JNK,but not MAPK,activation is associated with Fas-mediated apoptosis in human T cells

Fas is a cell surface molecule that is expressed on a wide array of cell types and triggers apoptosis. While in most situations Fas ligation activates programmed cell death, on resting T lymphocytes it can co-stimulate proliferation with the T cell receptor (TCR)/CD3 complex. This incongruity suggests that Fas may elicit signaling events that overlap with those used by proliferation cues. We observe that in the human T cell line Jurkat and in human peripheral blood lymphocytes, Fas stimulation does not signal by the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway or by increased intracellular calcium. Rather, Fas ligation strongly activates Jun kinase (JNK). This activity, as well as Fas-induced apoptosis, is blocked by increased levels of cAMP. The balance between proliferation and apoptosis by Fas triggering of T lymphocytes may therefore reflect a signaling ratio between TCR activation of the Ras/Raf-1/MAPK pathway versus JNK activation by Fas.     

Long-term proliferation and survival of in vitro-activated T cells is dependent on Interleukin-2 receptor signalling but not on the high-affinity IL-2R

Optimization of T‐cell‐activation protocols is an important prerequisite for the use of populations of activated, polyclonal T cells for immunotherapeutic purposes. This study compares two activation protocols. Following initial CD3/CD28 activation, naïve and memory subsets of CD8⁺ and CD4⁺ T cells were either repeatedly stimulated or maintained in medium containing interleukin‐2 (IL‐2). Initially, activation‐induced cell death (AICD) was observed in all subsets. After 2–3 days, death in the cultures maintained in IL‐2 only dropped dramatically, while live cells increased logarithmically. Despite intense proliferation, these cells lost the expression of CD25, the α chain of the IL‐2 receptor and CD71, the transferrin receptor. Functional blocking of CD25 caused minimal changes in proliferation and survival of these cells as long as IL‐2 was present in the medium. Blocking of CD25 in combination with the removal of IL‐2 caused rapid death of these cells. Restimulation every 3–4 days led to persistently high levels of AICD and lower live cell counts. Live cells maintained the expression of activation markers and a blastoid phenotype. Initial CD3/CD28 followed by maintenance in IL‐2 for 2–3 weeks seems to be the best in vitro T‐cell‐activation strategy. Signalling through the IL‐2 receptor is vital for these cells, despite their downregulation of CD25.     

Serotonin-1 receptor binding sites in the human basal ganglia are decreased in Huntington's chorea but not in Parkinson's disease: a quantitative in vitro autoradiography study

Serotonin-1 receptors were examined in post-mortem human brains, using quantitative in vitro autoradiography. [3H]Serotonin was used as a ligand. Serotonin-1 receptor subtypes were defined with 8-hydroxy-2-(di-n-propylamino)-tetralin and mesulergine. In the control human basal ganglia, the highest density of serotonin-1 binding sites was observed in both lateral and medial globus pallidus and substantia nigra reticulata. Lower densities were seen in the substantia nigra pars compacta, the nucleus accumbens, caudate and putamen. The majority of these serotonin-1 sites belonged to the serotonin-1D class. No significant alteration of the density and distribution of these sites was observed in Parkinson's disease brains. In contrast, a marked decrease in the density of the receptor binding was seen in the basal ganglia and the substantia nigra from patients dying with Huntington's disease. These results suggest that serotonin-1D receptors are expressed by cells intrinsic to the striatum which degenerate in Huntington's disease and project to the substantia nigra reticulata where these receptors are probably presynaptically localized. These observations in pathological human brains agree with the results of lesion studies in animal models and further support a role for serotoninergic mechanisms in movement control.     

Activation via the antigen receptor is impaired in T cells,but not in B cells from patients with common variable immunodeficiency

The patients included in this study belong to a subset of common variabie immunodeficiency (CVID) patients whose peripheral blood T cells have a T cell receptor (TCR)-mediated activation defect leading to impaired expression of the interleukin (IL)-2 gene upon stimulation with recall antigens (tetanus toxoid, Escherichia coli) or superantigens (staphylococcal enterotoxins). In the present report we demonstrate that the patients' peripheral blood T cells failed to generate the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) following stimulation with superantigen or mAb specific for the monomorphic region of the TCR β-chain. Patients' T cell lines were also impaired in generating Ins(1,4,5)P₃ when stimulated with tetanus toxoid-pulsed autologous monocytes. Addition of a second or third co-stimulatory signal provided by recombinant IL-2, CD28 or both had no effect on the Ins(1,4,5)P₃ formation of the patients' antigen-driven T cell lines. The T cell activation defect, however, was not absolute, as Ins(1,4,5)P₃ formation in the patients' T cells after phytohemagglutinin or aluminium fluoride stimulation was normal. The impairment in signal transduction via the T cell antigen receptor was limited to the patients' T cells, as no activation defect after ligation of surface immunoglobulin, the antigen receptor on B cells, could be detected.     

GLUT11, but not GLUT8 or GLUT12, is expressed in human skeletal muscle in a fibre type-specific pattern

Nine novel sugar transporter-like proteins have been discovered in the past 5 years. The mRNA for three of these, the glucose transporters (GLUT) GLUT8, GLUT11 and GLUT12, have been detected in human skeletal muscle. In the present study, we examined the pattern of expression and localization of the GLUT isoforms 8, 11 and 12 in human skeletal muscle using an immunohistochemical approach. Biopsies of human skeletal muscle from sedentary or trained healthy adults, from fetal muscle (24 weeks of gestation), from obese type-2 diabetic subjects, and from patients suffering from polymyositis or amyotrophic lateral sclerosis (ALS) were studied. GLUT8 and 12 immunoreactivity was below detection level in both developing and adult muscle fibres. GLUT11 immunoreactivity, however, was present in slow-twitch muscle fibres, but not in fast twitch fibres. Since, in contrast, GLUT4 was expressed in all investigated muscle fibres, the pattern of expression of GLUT11 differs from that of GLUT4, suggesting a specialized function for GLUT11 with a regulation independent from that of GLUT4. Obesity, type-2 diabetes, training, conditions of de- and reinnervation (ALS) and regeneration (polymyositis) failed to induce GLUT8 or -12 expression. Likewise, the fibre type-dependent pattern of GLUT11 immunoreactivity was unaltered. However, some slow muscle fibres lose their GLUT11 immunoreactivity under regeneration. Our results indicate that GLUT11 immunoreactivity, in contrast to that of GLUT4, is expressed exclusively in slow-twitch muscle fibres and is unaffected by physiological and pathophysiological conditions except in primary myopathy. GLUT8 and GLUT12 do not appear to be of importance in human muscle under physiological and pathophysiological conditions.