Lymphocyte Cell Surface Research Articles

Сіальованість глікопротеїнів і рівень експресії нейрамінідази NEU1 та сіалілтрансферази ST6GAL1 у лімфоцитах хворих на еритремію

We studied the levels of lymphocyte surface-associated sialylglycans and the expression of neuraminidase NEU1 and sialyltransferase ST6GAL1 genes in lymphocytes in erythremia patients and healthy donors as well as the levels of sialic acids in plasma and sialylation of alpha-acid glycoprotein and fibronectin. Moreover, we also investigated the type of sialic acids binding with its glycans using sialospecific lectins MAA and SNA. fibronectin protein in lymphocytes and its cell surface in erythremia disease as compared to healthy donors. It was shown that the levels of free sialic acids and neuraminidase activity in plasma are increased in erythremia disease as compared to healthy donors; however, MAA-II-binding activity of tested glycoproteins is decreased, fibronectin-1 mRNA expression in lymphocytes is increased in patients with erythremia. The decreasing of plasma fibronectin concentration and its heparin-binding activity as well as increasing of lymphocyte content with surface-associated and intracellular fibronectin were revealed in erythremia disease in comparison with healthy donors. Positive correlation between plasma fibronectin level and its heparin-binding activity and negative correlation between plasma fibronectin level and quantity of lymphocytes which express fibronectin inside the cell and on cell surface was detected. Enhanced levels of α2,3- and α2,6-linked residues of glycocojugates were detected on lymphocyte cell surface in erythremia disease using sialospecific lectins and flow cytometer as well as fluorescent confocal microscope. The level of NEU1 and ST6GAL1 mRNA expressions is significantly increased in lymphocytes in erythremia disease. Results of this study are clarified the mechanisms of disturbed in erythremia disease glycobiological processes and may therefore present new approaches for therapeutic opportunities.

40IN - Immunotherapy for Prostate Cancer?

ABSTRACT Sipuleucel-T is a cell based therapy for metastatic, castration-resistant prostate cancer (mCRPC) that was approved by the U.S. FDA in 2010. Data from randomized Phase III trials of this agent, as well as Phase II data from second active immunotherapy for prostate cancer (ProstVac VF) provide strong evidence that prostate cancer may be sensitive to immune pressure, although objective responses (OR's) are quite rare. An alternative approach to immunotherapy focuses on a series of molecules known as immune checkpoints, which are expressed on the cell surface of tumor-infiltrating lymphocytes in multiple tumor types. Blocking immune checkpoint molecules with monoclonal antibodies can promote T cell activation and lead to OR's in patients with melanoma, kidney cancer and NSCLC. Immune checkpoint blockade has been less effective in prostate cancer, with a recent randomized Phase III study, anti-CTLA-4 failing to meet its primary (OS) endpoint. The reasons for this are not well-understood, but may involve a differential sensitivity of visceral versus non-visceral metastases to immunotherapy. Ongoing studies in our laboratory are centered around a more precise understanding of the molecular phenotype of the T cells that infiltrate high-grade tumors, based on the hypothesis that checkpoint molecules involved in attenuating an ant-tumor immune response in prostate cancer might be different than those involved in other tumor types. Disclosure: C. Drake: Paid consultant: Bristol Myers Squibb, Compugen, Dendreon, ImmunExcite, Janssen, Pfizer, Roche-Genentech, Sanofi Aventis. SAB: Compugen.

Analysis of Toll-Like Receptors, iNOS and Cytokine Profiles in Patients with Pulmonary Tuberculosis during Anti-Tuberculosis Treatment

Toll-like receptors (TLRs) play an important role in mycobacterial infection, although little is known about the roles of these receptors, cytokines and nitric oxide during anti-tuberculosis treatment. Our objective was to evaluate the mRNA and cell surface expression of TLR2 and TLR4; inducible nitric oxide synthase (iNOS) expression; and cytokine Th1, Th2 and Th17 profiles in pulmonary tuberculosis patients at different time points of anti-tuberculosis treatment. Peripheral blood mononuclear cells (PBMCs) were obtained from PPD+ healthy controls and from patients receiving anti-tuberculosis treatment. Gene expression quantification was performed by qPCR, cell surface expression was assessed using flow cytometry, and cytokine quantification was conducted using the CBA technique. The treated patients presented higher gene expression and higher numbers of receptors on the cell surface of lymphocytes and monocytes than did control individuals. IL-12 and IFN-γ levels increased after the start of treatment, whereas TNF-α levels were reduced. TGF-β presented the highest levels during treatment. IL-10 and IL-17 expression and production tended to increase during treatment. iNOS gene expression was reduced throughout treatment in patients. Our results suggest that anti-tuberculosis treatment modulates the immune response, inducing an increase in the expression of TLRs and pro- and anti-inflammatory cytokines to combat bacteria and reduce the inflammatory process.

Ultrastructural analysis of nanogold-labeled cell surface microvilli in liquid by atmospheric scanning electron microscopy and their relevance in cell adhesion.

The adhesion of leukocytes circulating in the blood to vascular endothelium is critical for their trafficking in the vasculature, and CD44 is an important cell surface receptor for rolling adhesion. In this study, we demonstrate the correlative observation of CD44 distribution at the lymphocyte cell surface in liquid by fluorescence optical microscopy and immuno-electron microscopy using an atmospheric scanning electron microscope (ASEM). The ultrastructure of the cell surface was clearly imaged by ASEM using positively charged Nanogold particles. ASEM analysis demonstrated microvilli projections around the cell surface and the localization of CD44 on the microvilli. Treatment of cells with cytochalasin D resulted in a loss of the microvilli projections and concomitantly abrogated CD44-mediated adhesion to its ligand hyaluronan. These results suggest the functional relevance of microvilli in CD44-mediated rolling adhesion under shear flow.

Dysregulation of the antigen-induced NF-κB signaling pathway in the development of human B-cells lymphomas

The adaptive immune response is initiated when microbial or tumor-specific molecules are recognized by antigen receptors present at lymphocyte cell surface. Engagement of these immunoreceptors induces signaling cascades that ultimately activate the transcription factors NF-κB (Nuclear Factor-κB) and NFAT (nuclear factor of activated T-cells) responsible for the establishment of gene expression programs controlling the stimulation, proliferation and survival of activated lymphocytes. Cloning the products of three distinct translocation events found in lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) led to the identification and characterization of a novel NF-κB-activating complex following antigenic stimulation composed by the ARMA1, BCL10, and MALT1 proteins and called the CBM complex. Since the identification of this complex, other regulatory components were discovered which greatly improve our understanding of this signaling pathways. Interestingly, CBM complex activity is not only altered in MALT lymphomas but also in a subtype activated B cell-like (ABC) of diffuse large B-cell (DLBCL) lymphoma. In this review, we described the key players involved in antigen-induced NF-κB activation and cover the recent advances in the molecular mechanisms that are responsible for the regulation of the components of the CBM complex. Understanding these mechanisms is critical for the elucidation of the role of this NF-κB signaling network in both normal and pathologic conditions and we described how deregulation of this network leads to the uncontrolled activation of NF-κB and development of two particular subtypes of human B cell lymphomas: the MALT and the DLBCL lymphomas.

The Use of Nanomechanic Sensor for Studies of Morphofunctional Properties of Lymphocytes from Healthy Donors and Patients with Chronic Lymphoblastic Leukemia

Tapping mode-atomic force microscopy and force spectroscopy were used for studies of the topography of the cell surface and elastic properties of lymphocytes from healthy donors and patients with chronic lymphoblastic leukemia. It was demonstrated that the decrease in lymphocytes stiffness in patients with chronic lymphoblastic leukemia by 51.4% (p<0.05) was accompanied by spatial modification of the cell surface, in particular, increase in the number of globular protrusions and depressions by 247 and 122%, respectively (p<0.05), in comparison with normal lymphocytes.

Fingolimod

Oral fingolimod (Gilenya™), a sphingosine 1-phosphate (S1P) receptor agonist, is the first oral agent and the first in a novel class of disease-modifying therapies (DMTs) to be approved for use in the US for the treatment of relapsing forms of multiple sclerosis (MS). In the EU, fingolimod is approved for use as a single-agent DMT in selected patients with highly-active, relapsing-remitting (RR) MS. This article reviews the pharmacological properties and clinical use of the drug in patients with RRMS. Fingolimod is rapidly converted in vivo to the active moiety S-fingolimod-phosphate, which binds with high affinity to S1P receptors, thereby sequestering lymphocytes in the lymph nodes and preventing their egress into the peripheral circulation. As a consequence, there is a reduction in the infiltration of autoaggressive lymphocytes into the CNS. Fingolimod-phosphate also acts as a functional antagonist, as its binding to S1P receptors results in their internalization and degradation, thereby downregulating S1P receptors on the lymphocyte cell surface. Since fingolimod crosses the blood : brain barrier, it also potentially acts at S1P receptors on neural cells in the CNS to mitigate neuropathological processes associated with MS. In large multinational trials in adult patients with RRMS, oral fingolimod 0.5 mg/day was more effective than oral placebo (FREEDOMS) and recommended dosages of intramuscular interferon-β (IFNβ)-1a (TRANSFORMS) in reducing the annualized relapse rate and was also generally more effective at slowing progression of neurological disability and at reducing the burden and activity of disease. Fingolimod was generally well tolerated in these trials of up to 2 years' duration, with most adverse events being manageable and of mild to moderate severity; there were two deaths from opportunistic infections, albeit these occurred with fingolimod 1.25 mg/day (higher than the recommended dosage). Limited long-term data indicated that no new safety concerns had arisen after 5 years of fingolimod treatment. However, further clinical experience is required to fully determine the long-term safety profile of fingolimod, particularly with regard to any potentially serious or life-threatening adverse events. In the absence of robust pharmacoeconomic studies and of head-to-head trials comparing fingolimod with other formulations of IFNβ and glatiramer acetate, the relative position of fingolimod with respect to other DMTs remains to be fully determined. In the meantime, given its convenient once-daily oral treatment regimen and better efficacy than intramuscular IFNβ-1a, fingolimod is a valuable emerging option for the treatment of adult patients with relapsing forms of MS.

Antiphase signalling in the neuroendocrine-immune system in healthy humans

Background Any quantity varying in the spatial-temporal dimension may be considered as a signal. Human lymphocyte cell surface molecules and subsets present circadian variation and this variation may represent a kind of signalling in the neuroendocrine-immune system. We have analyzed the dynamics of variation of specific lymphocyte subsets in healthy humans. Subjects and methods In our study, lymphocyte subpopulation analyses were performed and cortisol, melatonin, GH and TSH serum levels were measured on blood samples collected every 4 h for 24 hours from eleven healthy men, ages 35–53 years (mean = 44 ± 6 SD). Results A clear circadian rhythm was validated for CD8 and cortisol with acrophase during the day and for CD3, CD4, melatonin, GH and TSH with acrophase at night. Cross-correlation showed that CD3 correlated positively with CD4 ( ρ = 0.67, P < 0.05) and negatively with CD8 ( ρ = −0.41, P < 0.05), CD4 correlated positively with melatonin ( ρ = 0.90, P < 0.05), GH ( ρ = 0.92, P < 0.05) and TSH ( ρ = 0.71, P < 0.05), negatively with CD8 ( ρ = −0.90, P < 0.05) and cortisol ( ρ = −0.18, P < 0.05), CD8 correlated positively with cortisol ( ρ = 0.38, P < 0.05). Discussion The different profiles of nyctohemeral changes of lymphocyte cell surface molecules and specific lymphocyte subsets realize different relationships with the neuroendocrine hormones and might represent a way of signal transmission among the multiple components of the neuroendocrine-immune system.

Acute dengue virus 2 infection in Gabonese patients is associated with an early innate immune response, including strong interferon alpha production

BackgroundDengue is now a leading cause of morbidity and mortality throughout the tropics. We conducted the first ex vivo study of dengue fever (DF) in African patients infected during the first Gabonese dengue virus 2 (DENV-2) outbreak in 2007, in order to investigate cytokine production, including the antiviral cytokine IFN-α, reported to be a potent inhibitor of DENV replication in vitro.MethodsLevels of 50 cytokines, chemokines and growth factors were measured in plasma from 36 patients with DENV-2 infection, and in uninfected controls, using Luminex multiplex technology. The results were interpreted according to the day of sampling after symptom onset. PBMC from six patients were also studied for T lymphocyte cell surface marker expression by flow cytometry.ResultsAcute DENV-2 infection elicited high levels of several pro-inflammatory cytokines (IL-6 and IL-17), chemokines (MIF, RANTES, IP-10 and MCP-1) and growth factors (G-CSF, GM-CSF and VEGF-A). We also observed high levels of IFN-α for the first time in adult DF patients, and CD4+ and CD8+ T cell activation at symptom onset.ConclusionAcute DENV-2 infection in African patients elicits a strong innate response involving IFN-α production, as well as an adaptive immune response.

Therapeutic effects of B and T lymphocyte attenuator extracellular domain and heat shock protein 70 antigen peptide on cervical cancer in mouse model

To investigate the synergistic therapy effects of B and T lymphocyte attenuator (BTLA) extracellular domain in combination with heat shock protein 70 (HSP70)-TC-1 antigen peptide complex on the mouse model of cervical cancer and the related immunological mechanisms. (1) Detecting the BTLA and herpesvirus entry mediator (HVEM) gene expression in the tumor microenvironment after C57BL/6 mice were inoculated with TC-1 tumor cells by realtime PCR; BTLA, HVEM expression on tumor infiltrating lymphocytes cell surface were detected by flow cytometry (fluorescence intensity). (2) According to different treatments, tumor-bearing mice were divided into 5 groups, which was injected with pcDNA3.1 (empty vector plasmid as control), psBTLA (vector plasmid which expresses BTLA extracellular domain), HSP70 (HSP70-TC-1 cell peptide complex), HSP70 + pcDNA3.1 or HSP70 + psBTLA, respectively. The weight of tumor was recorded. The expression of immunoregulatory genes in tumor microenvironment were detected. The change of lymphocyte amount and cytotoxicity were detected too; lymphocyte proliferation activity was measured by tritium thymidine incorporation assay; the concentration of interleukin (IL)2 and interferon-γ (IFN-γ) in supernatants of spleen lymphocyte were measured by enzyme-linked immunosorbent assay (ELISA). (1) BTLA gene expression was gradually increased after tumor cells inoculation. The highest expression level was 2.83 ± 0.35 at 14th day, which had statistical significance difference with the 7th day expression of 1.66 ± 0.25 (P < 0.05). While HVEM mRNA expression did not change significantly (P > 0.05). The 7th and 14th day after TC-1 cells inoculation, the average fluorescence intensity of BTLA expression on the surface of tumor infiltrating lymphocytes was 33.5 and 51.8, respectively, in which there was statistically significant difference (P < 0.05); while the difference of HVEM expression was not statistically significant (57.2 vs 49.3, P > 0.05). (2) The 28th day after inoculation, tumor inhibition rate of HSP70 + psBTLA group was 88%, which was significantly higher than other treatment groups (P < 0.05). The 28th day after TC-1 cells inoculation, combination therapy not only promoted IFN-γ and IL-2 gene (3.12 ± 0.71, 3.20 ± 0.62) expression but also reduced transforming growth factor-β (TGF-β), Foxp3 and IL-10 expression (0.25 ± 0.03, 0.19 ± 0.03, 0.31 ± 0.04; P < 0.05). It also promoted CD₈(+) T lymphocyte infiltration (52 ± 6)/high power field, cytotoxicity (65.5 ± 2.4)%, proliferation (15.0 × 10³ cpm) and cytokine IL-2, IFN-γ secretion (824 ± 51), (1096 ± 112) pg/ml, which were all significantly higher than other groups (P < 0.05). The effect of immunotherapy on tumor can be augmented by the combination of psBTLA which expresses extracellular domain of BTLA and HSP70-TC-1 tumor antigen peptide complex, which could improve the expression of the related immunoregulatory genes to establish a much better microenvironment in favor of anti-tumor immune response against the mice model of the cervix carcinoma.

Lymphocyte and monocyte flow cytometry immunophenotyping as a diagnostic tool in uncharacteristic inflammatory disorders

BackgroundPatients with uncharacteristic inflammatory symptoms such as long-standing fatigue or pain, or a prolonged fever, constitute a diagnostic and therapeutic challenge. The aim of the present study was to determine if an extended immunophenotyping of lymphocytes and monocytes including activation markers can define disease-specific patterns, and thus provide valuable diagnostic information for these patients.MethodsWhole blood from patients with gram-negative bacteraemia, neuroborreliosis, tuberculosis, acute mononucleosis, influenza or a mixed connective tissue disorders, as diagnosed by routine culture and serology techniques was analysed for lymphocyte and monocyte cell surface markers using a no-wash, no-lyse protocol for multi-colour flow cytometry method. The immunophenotyping included the activation markers HLA-DR and CD40. Plasma levels of soluble TNF alpha receptors were analysed by ELISA.ResultsAn informative pattern was obtained by combining two of the analysed parameters: (i), the fractions of HLA-DR-expressing CD4+ T cells and CD8+ T cells, respectively, and (ii), the level of CD40 on CD14+ CD16- monocytes. Patients infected with gram-negative bacteria or EBV showed a marked increase in monocyte CD40, while this effect was less pronounced for tuberculosis, borrelia and influenza. The bacterial agents could be distinguished from the viral agents by the T cell result; CD4+ T cells reacting in bacterial infection, and the CD8+ T cells dominating for the viruses. Patients with mixed connective tissue disorders also showed increased activation, but with similar engagement of CD4+ and CD8+ T cells. Analysis of soluble TNF alpha receptors was less informative due to a large inter-individual variation.ConclusionImmunophenotyping including the combination of the fractions of HLA-DR expressing T cell subpopulations with the level of CD40 on monocytes produces an informative pattern, differentiating between infections of bacterial and viral origin. Furthermore, a quantitative analysis of these parameters revealed the novel finding of characteristic patterns indicating a subacute bacterial infection, such as borreliosis or tuberculosis, or a mixed connective tissue disorder. The employed flow cytometric method is suitable for clinical diagnostic laboratories, and may help in the assessment of patients with uncharacteristic inflammatory symptoms.

Tim3 Is Upregulated and Protective in Nephrotoxic Serum Nephritis

T cell immunoglobulin and mucin protein-3 (Tim3) is mainly expressed on the cell surface of T-helper lymphocytes (T(H)) that negatively regulates T(H)-type 1 (T(H)-1) responses. Because blockade of Tim3 aggravates disease activity in T(H)-1-dependent diseases, we investigated whether Tim3 is involved in the pathogenesis of the T(H)-1-dependent nephrotoxic nephritis (NTS). We first evaluated Tim3 expression in mice after induction of nephrotoxic serum nephritis (NTS) and then studied the effects of anti-Tim3 treatment toward the course of NTS for up to seven days. Whereas Tim3 expression was undetectable in control mice, we found significantly increased Tim3 expression in kidneys, but not in draining lymph nodes, at one, four, and eight weeks after induction of NTS. Tim3-expressing cells that infiltrated kidneys of mice subjected to NTS turned out to be CD4(+) T cells rather than CD8(+) cytotoxic T cells and dendritic cells. Administration of a blocking anti-Tim3 antibody aggravated nephritis as shown by significantly increased albuminuria, respective histological changes, and increased expression of the kidney injury molecule lipocalin-2. In parallel, an increase of infiltrating T cells, macrophages, and macrophage pro-inflammatory cytokine formation as well as increased proliferation and apoptosis in kidneys of anti-Tim3-treated mice was detected. Together, we provide the first evidence that Tim3 is up-regulated in kidneys in NTS and that Tim3 exerts protective roles in the course of disease.

Constitutively overexpressed 21 kDa protein in Hodgkin lymphoma and aggressive non-Hodgkin lymphomas identified as cytochrome B5b (CYB5B)

BackgroundWe have previously reported a novel constitutively overexpressed 21 kDa protein in Hodgkin Lymphoma (HL) and aggressive Non-Hodgkin Lymphomas (NHL). The objective of the current study was to 1) identify this protein using two independent methods, 2) study the expression of the protein and its encoding mRNA in reactive lymph nodes, normal lymphocytes and CD34+ bone marrow precursor cells, 3) analyse patterns of expression of the protein in tissue microarrays assembled from a large number of diagnostic clinical biopsies from patients with HL, and 4) determine the copy number variation and mutation status of the encoding gene in HL cell lines.ResultsPeptide sequencing by LC-MS/MS and protein identification by protein array screening identified a single protein, CYB5B. No mutations were detected in the CYB5B gene in HL cell lines. Quantitative PCR showed CYB5B gene expression was increased in HL and NHL cell lines. Array CGH using a submegabase resolution tiling array revealed gains in the CYB5B locus in HL cell lines KMH2 and L428. Membrane expression was seen in Reed-Sternberg cells in clinical biopsies from patients with HL but not in reactive lymph nodes. Bone marrow CD34+ precursor cells were CYB5B negative on the cell surface. RT-PCR assays of RNA extracted from T and B cell enriched fractions obtained from normal peripheral blood mononuclear cells, reactive lymph nodes, tonsils and normal bone marrow samples showed no evidence of increased mRNA levels of CYB5B in comparison to housekeeping gene GAPDH.ConclusionsThe 21 kDa protein overexpressed in HL and aggressive NHL is identical to CYB5B. CYB5B gene expression is increased in a subset of HL and NHL cell lines tested. This is associated with CYB5B gene amplification in HL cell lines KMH2 and L428. CYB5B may be a potential target for antibody-based therapy of HL and aggressive NHL as although cytoplasmic expression is present in reactive lymphocytes, it is not expressed on the cell surface of non-neoplastic lymphocytes or bone marrow precursor cells.

C-Cbl mediated ubiquitylation and regulation of cell surface exposure of CD5

Downregulation of cell surface receptors is an important process aimed at attenuation or termination of receptor signaling. c-Cbl role in the process is thought to be initial ubiquitylation of the receptors targeted for degradation and assembly of internalization complexes consisting of several other proteins. c-Cbl seems to be present during the whole process of vesicle sorting after internalization. However, there are very few receptor molecules so far like EGFR being proven to be regulated by c-Cbl. It is known that a level of CD5 on mouse c-Cbl−/− thymocytes is upregulated in comparison to wild type cells. The mechanism leading to the upregulation is unknown. We show that CD5 is ubiquitylated in Jurkat-TAg cells and in mouse thymocytes and that the ubiquitylation is c-Cbl dependent. We also show that amount of CD5 associated with lysosomal marker LAMP-1 after stimulation is significantly lower in c-Cbl −/− thymocytes. CD5 mRNA level did not differ significantly between c-Cbl −/− and wild type thymocytes. We conclude that CD5 is ubiquitylated; the ubiquitylation is mediated by c-Cbl; CD5 level on a T lymphocyte cell surface is regulated by ubiquitylation and targeting to lysosomes.

Static Magnetic Field Interferes with the Physiological Removal of Circulating Apoptotic Lymphocytes

In various cellular systems, including cells of the immune system, a number of biological efiects induced by static magnetic flelds (SMFs) have been reported and difierent mechanisms have been proposed to explain these efiects. Although a number of theoretical mod- els have been proposed, the variety of experimental conditions (intensity, frequency and time windows of the flelds, difiering characteristics of the materials used- cell type, age, treatment) makes contradictory the data present in the literature and the possibility of the replication of the experiments. However, (S)MFs have been reported to perturb distribution of membrane proteins and sugars, cytoskeleton and trans-membrane ∞uxes of difierent ions, especially calcium (Ca 2+ )i. In turn, these alterations could interfere with speciflc physiological activies, like phagocytosis, which are based on receptors, cytoskeleton elements and motor proteins. In a previous work we found that, sinusoidal liver cells quickly recognized and engulfed human lymphocytes exposed for up to 72h to 6mT SMF, by using the same receptors that mediate the clearance of apoptotic cells. Thus, aim of the present work has been to decipher the modiflcations exerted by the SMF on lymphocytes and/or on the process of phagocytose. We analysed the cell surfaces of normal and apoptotic human lymphocytes in the presence or absence of 6mT SMF by immunocyto- chemistry and biochemistry assays. SMF increases, in a time-dependent way, the expression of GD3 ganglyoside and cholesterol on the plasma membrane of normal lymphocytes and prevents GD3 removal on apoptotic-induced cells. Lipid peroxidation of plasma membrane was observed soon after lymphocytes induction of apoptosis and after 72h of SMF exposure. The recognition and the engulfment of the control and apoptotic lymphocytes is modifled by SMF exposure. In fact, normal exposed lymphocytes are recognized by the liver sinusoids at the same extent of the apoptotic non exposed cells. Conversely, the exposure to SMF promoted the binding but delayed the engulfment of apoptotic lymphocytes in in situ as well as in in vitro phagocytose assays. Further studies will clarify the eventual implication of SMF on human health.

N- and O-Glycans Modulate Galectin-1 Binding, CD45 Signaling, and T Cell Death

Galectin-1, a beta-galactoside-binding protein highly expressed in the thymus, induces apoptosis of specific thymocyte subsets and activated T cells. Galectin-1 binds to N- and O-glycans on several glycoprotein receptors, including CD7, CD43, and CD45. Here we show that galectin-1 signaling through CD45, which carries both N- and O-glycans, is regulated by CD45 isoform expression, core 2 O-glycan formation and the balance of N-glycan sialylation. Regulation of galectin-1 T cell death by O-glycans is mediated through CD45 phosphatase activity. While galectin-1 signaling in cells expressing low molecular weight isoforms of CD45 requires expression of core 2 O-glycans (high affinity ligands for galectin-1), galectin-1 signaling in cells expressing a high molecular weight isoform of CD45 does not require core 2 O-glycans, suggesting that a larger amount of core 1 O-glycans (low affinity ligands for galectin-1) is sufficient to overcome lack of core 2 O-glycans. Furthermore, regulation of galectin-1 signaling by alpha2,6-sialylation of N-glycans is not solely dependent on CD45 phosphatase activity and can be modulated by the relative expression of enzymes that attach sialic acid in an alpha2,6- or alpha2,3-linkage. Thus, N- and O-glycans modulate galectin-1 T cell death by distinct mechanisms, and different glycosylation events can render thymocytes susceptible or resistant to galectin-1.

Investigation of the sensitising and cross-sensitising potential of textile dyes and β-lactam antibiotics using a biphasic mice local lymph node assay

We used a modified protocol of the murine local lymph node assay (LLNA) to study the cross-sensitising potential of (a) textile dye disperse yellow 3 and its metabolite 2-amino-p-cresol, (b) two antibiotics, penicillin G and cefotiam. The test substances were applied in a biphasic manner, i.e. first on the shaved skin of the back followed by application on the dorsal side of the ears after 2 weeks. The end-points analysed included thickness and weight of an ear-biopsy, weight and cell number of the draining lymph node, and lymphocyte cell surface markers analysed by flow-cytometry. Disperse yellow 3 and its metabolite significantly altered the various end-points at both the tested concentrations (0.5 and 1%), thus demonstrating the sensitising potential of the two substances. The cross-sensitisation study showed significant modulation in the tested variables in the treated group as compared to the control, signifying cross-sensitisation potential of the two substances. Penicillin G and cefotiam showed significant changes in various end-points, pointing towards their sensitising potential. However, even at 50% concentration of the beta-lactams no significant change in any end-point indicating absence of cross-reactivity of the antibiotics was noticed. We conclude that a biphasic, modified protocol of the LLNA is a suitable approach to test for a cross-reactivity potential of two related compounds.

CD5 sweetens lymphocyte responses

The cell walls of pathogenic and commensal bacteria and fungi contain a variety of carbohydrate-containing moieties, not produced by animals. The presence of these lipopolysaccharides (LPSs), lipoteichoic acids (LTAs), peptidoglycans (PGNs), and zymosans in host tissues thus alerts the host to invasive bacterial or fungal infections. Several families of pattern recognition receptors (PRRs) expressed by epithelial and phagocytic cells contribute to recognition of microbial cell wall components. These PRRs include toll-like receptors (TLRs), nod-like receptors (NLRs), and scavenger receptors. Cell surface TLRs and cytosolic NLRs respond to microbial components by eliciting production of cytokines and chemokines that activate or amplify host inflammatory and immune responses. In contrast, at least some members of the scavenger-receptor cysteine-rich (SRCR) superfamily contribute more to the resolution of inflammation. In this issue of PNAS, a lymphocyte-expressed SRCR, CD5, is shown to bind the fungal polysaccharide β-glucan (1). Together with a recent study on CD6 (2), this work shows that SRCRs expressed on the lymphocyte cell surface are PRRs that detect diverse microbe-derived carbohydrates. Moreover, previous data implicating CD5 in negative regulation of lymphocyte antigen receptor signaling suggest that ligation of these PRRs with microbial components may profoundly influence lymphocyte activation and autoimmunity (Fig. 1).

Decreased Expression of T Lymphocyte Co-Stimulatory Molecule CD26 on Invariant Natural Killer T Cells in Systemic Lupus Erythematosus

CD26, a T cell co-stimulatory molecule and dipeptidyl peptidase IV for the degradation of interferon-γ-induced chemokine, participates in multiple immunopathological roles in leukocyte homing and inflammation. Decreased circulating concentration of soluble (s)CD26 in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis and murine model of arthritis and encephalomyelitis have been reported. In the present study, the plasma concentration of sCD26 and chemokines, and cell surface expression of CD26 on monocytes, CD4+T lymphocytes, CD8+T lymphocytes, CD19+B lymphocytes and invariant natural killer T (iNKT) lymphocytes were analyzed using ELISA and flow cytometry, respectively, in 23 SLE patients and 14 sex- and age-matched control subjects. Although there was no significant difference between plasma concentrations of soluble CD26 in SLE patients with controls (p > 0.05), there was significant elevated Th1 chemokines CXCL10 and CXCL9 but not Th2 chemokine CCL2, and down-regulation in iNKT lymphocytes number and cell surface expression of CD26 on CD4+T and iNKT lymphocytes of SLE patients compared with controls (all p < 0.05). Decreased circulating number of iNKT cells and CD26 on iNKT cells can be important for the immunopathogenesis by exacerbating Th1-related inflammation in SLE.

Dopamine as an immune-modulator between dendritic cells and T cells and the role of dopamine in the pathogenesis of Rheumatoid Arthritis

The nerve systems affect immune functions by releasing neurotransmitters through lymphocyte cell-surface receptors. A major neurotransmitter dopamine transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. There is wide evidence for a decreased risk of rheumatoid arthritis (RA) in patients with schizophrenia which is associated with the excessive stimulation of D2-like receptors by dopamine. However, the reason for the negative association between RA and schizophrenia is unknown. We previously demonstrated that dendritic cells (DCs) could synthesize and store dopamine, DC released dopamine to naive CD4 T cells upon DC-T cell interaction and affected helper T-cell differentiation. Because DCs have been proposed to play a pivotal role in the initiation and perpetuation of RA by presentation of arthritogenic antigens to T cells, we here assessed effects and functions of dopamine on immune cells during the pathogenesis of RA. In this paper, we overview the series of our research findings, and present the possibility of drug discovery which target at dopamine receptors.