Receptor Cross-linking Research Articles

Contributors to HMGB1 Release by Urothelial Carcinoma Cells in Response to Bacillus Calmette-Guérin

Our group previously noted that HMGB1 release by urothelial carcinoma cells occurs as a consequence of bacillus Calmette-Guérin induced nonapoptotic cell death. Additional studies demonstrated that HMGB1 release in response to bacillus Calmette-Guérin is required for the invivo tumor response to bacillus Calmette-Guérin. We evaluated the steps required for HMGB1 release by human urothelial carcinoma cells in response to bacillus Calmette-Guérin exposure. We used the T24 and 253J human urothelial carcinoma cell lines. HMGB1 concentrations in cell culture supernatant with and without bacillus Calmette-Guérin treatment served as the principal end point to assess the role of potentially involved variables. Specific techniques were used to determine the role of α5β1 antigen receptor cross-linking, TLR signaling, inducible nitric oxide synthase expression/nitric oxide production, p21 expression, and bacillus Calmette-Guérin adherence, internalization and viability. Cross-linking of α5β1 integrin or signaling through TLR2/4 did not contribute to HMGB1 release. Optimal HMGB1 release required bacillus Calmette-Guérin adherence and internalization. Bacillus Calmette-Guérin viability correlated with the magnitude of HMGB1 release. Inhibition of oxidative stress and p21 expression in response to bacillus Calmette-Guérin decreased the magnitude of HMGB1 release. Bacillus Calmette-Guérin induced nonapoptotic cell death and HMGB1 release occur as a consequence of a complex multistep process. Understanding the steps and mechanisms involved in the induced HMGB1 release would provide an opportunity for targeted strategies to improve bacillus Calmette-Guérin treatment efficacy.

586 CONTRIBUTORS TO HMGB1 RELEASE BY UROTHELIAL CARCINOMA CELLS IN RESPONSE TO BCG

You have accessJournal of UrologyBladder Cancer: Basic Research (I)1 Apr 2013586 CONTRIBUTORS TO HMGB1 RELEASE BY UROTHELIAL CARCINOMA CELLS IN RESPONSE TO BCG Guangjian Zhang, Fanghong Chen, Yanli Cao, Gopit Shah, and William See Guangjian ZhangGuangjian Zhang Milwaukee, WI More articles by this author , Fanghong ChenFanghong Chen Milwaukee, WI More articles by this author , Yanli CaoYanli Cao Milwaukee, WI More articles by this author , Gopit ShahGopit Shah Milwaukee, WI More articles by this author , and William SeeWilliam See Milwaukee, WI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1982AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent work by our group has demonstrated that the powerful chemokine and marker of non-apoptotic cell death, high molecular group box 1 (HMGB1), is released by a portion of urothelial carcinoma (UC) tumor cells following BCG exposure. Elevated levels of HMGB1 have been observed in the urine of patients following intravesical BCG therapy. Most importantly, in vivo in models have demonstrated that tumor cells engineered to have reduced HMGB1 release fail to respond to intravesical BCG administration. Given the apparent importance of HMGB1 release for in vivo anti-tumor activity, this study evaluated the role of specific elements of BCG/tumor cell interaction in contributing to maximal HMGB1 release. METHODS Two human UC cell lines, T24 and 253J, were employed. HMGB1 concentrations in cell culture supernatant, with and without BCG treatment, served as the principal end point to assess the role of potentially involved variables. Specific techniques were utilized to determine the role of α5β1 antigen receptor crosslinking, BCG adherence, BCG internalization, BCG viability, iNOS expression/NO production, and p21 expression in mediating HMGB1 release by UC cells in response to BCG. RESULTS Crosslinking of α5β1 integrin was insufficient to induce HMGB1 release. HMGB1 release by crosslinked cells was not significantly different than untreated controls (p > 0.5). Treatment of cells with the peptide GRDGS to block α5β1 integrin and inhibit BCG adherence significantly inhibited HMGB1 release (p<0.001). Inhibition of BCG internalization by pretreatment of cells with Cytochalasin B significantly decreased HMGB1 release relative to BCG alone (p < 0.0001). Both viable and heat killed BCG significantly increased HMGB1 release by UC cells relative to controls (p < 0.01) The use of heat killed BCG significantly reduced HMGB1 release to 50% of values observed in response to viable BCG (p<0.0001). HMGB1 release by iNOS inhibited cells was significantly reduced (p < 0.01). Inducible knockdown of p21 expression in response to BCG significantly reduced HMGB1 release following BCG treatment (p < 0.01). CONCLUSIONS BCG induced non-apoptotic cell death and HMGB1 release occurs as a consequence of a complex multi-step process. Optimal HMGB1 release requires the adherence and internalization of viable BCG, iNOS activity, and p21 expression. An understanding of the steps and mechanisms involved in BCG induced HMGB1 release affords an opportunity for targeted strategies to improve BCG treatment efficacy. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e239-e240 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guangjian Zhang Milwaukee, WI More articles by this author Fanghong Chen Milwaukee, WI More articles by this author Yanli Cao Milwaukee, WI More articles by this author Gopit Shah Milwaukee, WI More articles by this author William See Milwaukee, WI More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Granzyme D Is a Novel Murine Mast Cell Protease That Is Highly Induced by Multiple Pathways of Mast Cell Activation

Granzymes are serine proteases known mostly for their role in the induction of apoptosis. Granzymes A and B have been extensively studied, but relatively little is known about granzymes C to G and K to M. T cells, lymphohematopoietic stromal cells, and granulated metrial gland cells express granzyme D, but the function of granzyme D is unknown. Here we show that granzyme D is expressed by murine mast cells and that its level of expression correlates positively with the extent of mast cell maturation. Coculture of mast cells with live, Gram-positive bacteria caused a profound, Toll-like receptor 2 (TLR2)-dependent induction of granzyme D expression. Granzyme D expression was also induced by isolated bacterial cell wall components, including lipopolysaccharide (LPS) and peptidoglycan, and by stem cell factor, IgE receptor cross-linking, and calcium ionophore stimulation. Granzyme D was released into the medium in response to mast cell activation. Granzyme D induction was dependent on protein kinase C and nuclear factor of activated T cells (NFAT). Together, these findings identify granzyme D as a novel murine mast cell protease and implicate granzyme D in settings where mast cells are activated, such as bacterial infection and allergy.

Liposomes Decorated with Apo2L/TRAIL Overcome Chemoresistance of Human Hematologic Tumor Cells

Human Apo2-ligand/TRAIL is a member of the TNF cytokine superfamily capable of inducing apoptosis on tumor cells while sparing normal cells. Besides its antitumor activity, Apo2L/TRAIL is also implicated in immune regulation. Apo2L/TRAIL is stored inside activated T cells in cytoplasmic multivesicular bodies and is physiologically released to the extracellular medium inserted in the internal membrane vesicles, known as exosomes. In this study we have generated artificial lipid vesicles coated with bioactive Apo2L/TRAIL, which resemble natural exosomes, to analyze their apoptosis-inducing ability on cell lines from hematological tumors. We have tethered Apo2L/TRAIL to lipid vesicles by using a novel Ni(2+)-(N-5-amino-1-carboxylpentyl)-iminodiacetic acid, NTA)-containing liposomal system. This lipidic framework (LUVs-Apo2L/TRAIL) greatly improves Apo2L/TRAIL activity, decreasing by around 14-fold the LC50 on the T-cell leukemia Jurkat. This increase in bioactivity correlated with the greater ability of LUVs-Apo2L/TRAIL to induce caspase-3 activation and is probably due to the increase in local concentration of Apo2L/TRAIL, improving its receptor cross-linking efficiency. More important, liposome-bound Apo2L/TRAIL overcame the resistance to soluble recombinant Apo2L/TRAIL exhibited by tumor cell mutants overexpressing Bcl-xL or by a Bax and Bak-defective Jurkat cell mutant (Jurkat-shBak) and are also effective against other hematologic tumor cells. Jurkat-Bcl-xL and Jurkat-shBak cells are resistant to most chemotherapeutic drugs currently used in cancer treatment, and their sensitivity to LUVs-Apo2L/TRAIL could have potential clinical applications.

Bronchial Epithelial Cells From Asthmatic Patients Express FcεRIγ At Higher Levels During Rhinovirus Infection

Allergic asthmatics have reduced rhinovirus (HRV)-induced innate immune responses mediated by increased plasmacytoid dendritic cell (pDC) expression of the high-affinity IgE receptor, FcεRI through unclear mechanisms. Interestingly, the γ subunit of FcεRI conducts extracellular signals downstream of FcεRI and other immunoregulatory receptors on pDCs, with the common endpoint of reduced interferon production after receptor cross-linking. It is unknown whether FcεRIγ is expressed in bronchial epithelial cells (BECs).

Rituximab Provides Steroid-Sparing Therapy in New-Onset Chronic Graft-Versus-Host Disease

s / Biol Blood Marrow Transplant 19 (2013) S129eS149 S140 3.15 vs.9.34%; p1⁄40.045). As reported in chronic HIV viremia, expanded CD21B cells in cGVHD patients also expressed higher inhibitory receptors namely FCRL4, CD22 and CD85J, chemokine receptors CD11c, CXCR3 and lower CD62L and CCR7 compared with naive (CD27-CD21+CD19+) and classical memory CD27+CD21+CD19+) B cells. Moreover, B cells in patients with cGVHD had a lower proliferative potential following B-cell receptor crosslinking compared with B cells from patients with no GvHD or HC, a feature of exhaustion associated with high expression of inhibitory receptors. Finally, the frequencies of circulating CD21B cells correlated with cGvHD grade (r1⁄40.67; P 1⁄4 .002) but not with time from SCT (r1⁄40.28; P 1⁄4 .15). These data suggest that CD21B cells may play a role in the pathogenesis of cGVHD and could be used as as a marker of disease severity.

Structure of glutaraldehyde cross-linked ryanodine receptor.

The ryanodine receptor (RyR) family of calcium release channels plays a vital role in excitation-contraction coupling (ECC). Along with the dihydropyridine receptor (DHPR), calsequestrin, and several other smaller regulatory and adaptor proteins, RyRs form a large dynamic complex referred to as ECC machinery. Here we describe a simple cross-linking procedure that can be used to stabilize fragile components of the ECC machinery, for the purpose of structural elucidation by single particle cryo-electron microscopy (cryo-EM). As a model system, the complex of the FK506-binding protein (FKBP12) and RyR1 was used to test the cross-linking protocol. Glutaraldehyde fixation led to complete cross-linking of receptor-bound FKBP12 to RyR1, and also to extensive cross-linking of the four subunits comprising RyR to one another without compromising the RyR1 ultrastructure. FKBP12 cross-linked with RyR1 was visualized in 2D averages by single particle cryo-EM. Comparison of control RyR1 and cross-linked RyR1 3D reconstructions revealed minor conformational changes at the transmembrane assembly and at the cytoplasmic region. Intersubunit cross-linking enhanced [(3)H]ryanodine binding to RyR1. Based on our findings we propose that intersubunit cross-linking of RyR1 by glutaraldehyde induced RyR1 to adopt an open like conformation.

ADAMTS: Novel proteases expressed by activated mast cells

Here we show that mast cells (MCs) express the metalloproteases of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, and that ADAMTS expression is influenced by MC activation. Co-culture of MCs with live Gram-positive bacteria caused a profound induction of ADAMTS-9 and -6, as well as down-regulated expression of ADAMTS-5. Similar patterns were also seen after MC activation with calcium ionophore and by immunoglobulin E receptor crosslinking. Moreover, ADAMTS-5, -6 and -9 were all induced by activation of terminally differentiated murine peritoneal MCs and in a human MC line. ADAMTS-9 up-regulation in response to immunoglobulin E receptor crosslinking was strongly dependent on Gö6976-sensitive protein kinase C and partly dependent on nuclear factor of activated T cells and nuclear factor kappa-light-chain-enhancer of activated B cells, respectively. The expression of ADAMTS-5, -6 and -9 was closely linked to MC maturation, as shown by their strong induction during the differentiation of bone marrow precursor cells into mature MCs. ADAMTS family members have been shown to possess aggrecanase activity. Accordingly, MCs were shown to express aggrecanase activity. Finally, ADAMTS-5 protein was detected in MCs by immunocytochemistry. Taken together, the present study reveals ADAMTS expression by MCs and that MC activation regulates the expression of these proteases, thus implicating the ADAMTS family of proteases in MC function.

Polyunsaturated Free Fatty Acids Inhibit Coupling Between the Er Ca2+ Sensor STIM1 and the Ca2+ Channel Protein Orai1 Stimulated by Ige Receptor Crosslinking or Thapsigargin in a Process that Correlates with Disruption of Lipid Order

Polyunsaturated fatty acids (PUFAs) have been found to be effective inhibitors of cell signaling in numerous contexts. We find that acute addition of these PUFAs in micromolar concentrations, including linoleic acid, substantially inhibits Ca2+ responses in mast cells stimulated by antigen-mediated crosslinking of FceRI or by the SERCA inhibitor, thapsigargin. In addition to inhibiting store-operated Ca2+ entry, linoleic acid inhibits antigen-stimulated release of Ca2+ from intracellular stores and granule exocytosis. Using AcGFP-Orai1 and STIM1-mRFP to monitor stimulated coupling in COS7 cells by FRET in a fluorimetry assay, we find effective inhibition of this association by linoleic acid added either before or after stimulation by thapsigargin and ATP. Stearic acid, a saturated fatty acid of the same length as linoleic acid, does not inhibit FRET or Ca2+ signaling when added at the same concentrations. We showed previously that giant plasma membrane vesicles exhibit liquid order/liquid disorder phase separation at low temperatures, and we find that stearic acid enhances whereas linoleic acid prevents this phase separation. Our results suggest that PUFAs interfere with STIM1-Orai1 coupling by a mechanism that correlates with inhibition of membrane order heterogeneity in cells.

Activation/Inhibition of mast cells by supra-optimal antigen concentrations

Mast cells (MCs) are tissue resident cells of hemopoietic origin and are critically involved in allergic diseases. MCs bind IgE by means of their high-affinity receptor for IgE (FcεRI). The FcεRI belongs to a family of multi-chain immune recognition receptors and is activated by cross-linking in response to multivalent antigens (Ags)/allergens. Activation of the FcεRI results in immediate release of preformed granular substances (e.g. histamine, heparin, and proteases), generation of arachidonic acid metabolites, and production of pro-inflammatory cytokines. The FcεRI shows a remarkable, bell-shaped dose-response behavior with weak induction of effector responses at both low and high (so-called supra-optimal) Ag concentrations. This is significantly different from many other receptors, which reach a plateau phase in response to high ligand concentrations. To explain this unusual dose-response behavior of the FcεRI, scientists in the past have drawn parallels to so-called precipitin curves resulting from titration of Ag against a fixed concentration of antibody (Ab) in solution (a.k.a. Heidelberger curves). Thus, for high, supra-optimal Ag concentrations one could assume that every IgE-bound FcεRI formed a monovalent complex with “its own Ag”, thus resulting in marginal induction of effector functions due to absence of receptor cross-linking. However, this was never proven to be the case. More recently, careful studies of FcεRI activation and signaling events in MCs in response to supra-optimal Ag concentrations have suggested a molecular explanation for the descending part of this bell-shaped curve. It is obvious now that extensive FcεRI/IgE/Ag clusters are formed and inhibitory molecules and signalosomes are engaged in response to supra-optimal cross-linking (amongst them the Src family kinase Lyn and the inositol-5′-phosphatase SHIP1) and they actively down-regulate MC effector responses. Thus, the analysis of MC signaling triggered by supra-optimal crosslinking holds great potential for identifying novel targets for pharmacologic therapeutic intervention to benefit patients with acute and chronic allergic diseases.

Anti‐allergic action of anti‐malarial drug artesunate in experimental mast cell‐mediated anaphylactic models

Allergy is an acquired hypersensitivity reaction of the immune system mediated by cross-linking of allergen-specific IgE-bound high-affinity IgE receptors, leading to immediate mast cell degranulation. Artesunate is a semi-synthetic derivative of artemisinin, an active component of the medicinal plant Artemisia annua. Artesunate is a clinically effective anti-malarial drug and has recently been shown to attenuate allergic asthma in mouse models. This study investigated potential anti-allergic effects of artesunate in animal models of IgE-dependent anaphylaxis. Anti-allergic actions of artesunate were evaluated in passive cutaneous anaphylaxis and passive systemic anaphylaxis mouse models, and in ovalbumin-induced contraction of bronchial rings isolated from sensitized guinea pigs. Direct mast cell-stabilizing effect of artesunate was examined in RBL-2H3 mast cell line and in mature human cultured mast cells. Anti-allergic signaling mechanisms of action of artesunate in mast cells were also investigated. Artesunate prevented IgE-mediated cutaneous vascular hyperpermeability, hypothermia, elevation in plasma histamine level, and tracheal tissue mast cell degranulation in mice in a dose-dependent manner. In addition, artesunate suppressed ovalbumin-mediated guinea pig bronchial smooth muscle contraction. Furthermore, artesunate concentration-dependently blocked IgE-mediated degranulation of RBL-2H3 mast cells and human culture mast cells. Artesunate was found to inhibit IgE-induced Syk and PLCγ1 phosphorylation, production of IP(3) , and rise in cytosolic Ca(+2) level in mast cells. We report here for the first time that artesunate possesses anti-allergic activity by blocking IgE-induced mast cell degranulation, providing a foundation for developing artesunate for the treatment of allergic asthma and other mast cell-mediated allergic disorders.

Critical role of the IgM Fc receptor in IgM homeostasis, B-cell survival, and humoral immune responses

IgM antibodies have been known for decades to enhance humoral immune responses in an antigen-specific fashion. This enhancement has been thought to be dependent on complement activation by IgM-antigen complexes; however, recent genetic studies render this mechanism unlikely. Here, we describe a likely alternative explanation; mice lacking the recently identified Fc receptor for IgM (FcμR) on B cells produced significantly less antibody to protein antigen during both primary and memory responses. This immune deficiency was accompanied by impaired germinal center formation and decreased plasma and memory B-cell generation. FcμR did not affect steady-state B-cell survival but specifically enhanced the survival and proliferation induced by B-cell receptor cross-linking. Moreover, FcμR-deficient mice produced far more autoantibodies than control mice as they aged, suggesting that FcμR is also required for maintaining tolerance to self-antigens. Our results thus define a unique pathway mediated by the FcμR for regulating immunity and tolerance and suggest that IgM antibodies promote humoral immune responses to foreign antigen yet suppress autoantibody production through at least two pathways: complement activation and FcμR.

Polydatin (PD) inhibits IgE-mediated passive cutaneous anaphylaxis in mice by stabilizing mast cells through modulating Ca2 + mobilization

Mast cells play a key role in the pathogenesis of asthma and are a promising target for therapeutic intervention in asthma. This study investigated the effects of polydatin (PD), a resveratrol glucoside, on mast cell degranulation upon cross-linking of the high-affinity IgE receptors (FcεRI), as well as the anti-allergic activity of PD in vivo. Herein, we demonstrated that PD treatment for 30min suppressed FcεRI-mediated mast cell degranulation in a dose-dependent manner. Concomitantly, PD significantly decreased FcεRI-mediated Ca2+ increase in mast cells. The suppressive effects of PD on FcεRI-mediated Ca2+ increase were largely inhibited by using LaCl3 to block the Ca2+ release-activated Ca2+ channels (CRACs). Furthermore, PD significantly inhibited Ca2+ entry through CRACs evoked by thapsigargin (TG). Knocking down protein expression of Orai1, the pore-forming subunit of CRACs, significantly decreased PD suppression of FcεRI-induced intracellular Ca2+ influx and mast cell degranulation. In a mouse model of mast cell-dependent passive cutaneous anaphylaxis (PCA), in vivo PD administration suppressed mast cell degranulation and inhibited anaphylaxis. Taken together, our data indicate that PD stabilizes mast cells by suppressing FcεRI-induced Ca2+ mobilization mainly through inhibiting Ca2+ entry via CRACs, thus exerting a protective effect against PCA.

Structure of the haptoglobin–haemoglobin complex

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected β-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).

Interaction of TAPP adapter proteins with phosphatidylinositol (3,4)‐bisphosphate regulates B‐cell activation and autoantibody production

TAPP1 and TAPP2 (where TAPP is tandem PH domain containing protein) are dual PH domain adaptors that selectively bind PI(3,4)P2 (phosphatidylinositol (3,4)-bisphosphate). PI(3,4)P2 is a lipid messenger generated by phosphoinositide 3-kinase (PI3K) and SHIP, both of which are critical regulators of B-cell activation. To determine the functional role of TAPP-PI(3,4)P2 interactions, we utilized a double knock-in (KI) mouse bearing mutations within the PI-binding pocket of both TAPP1 and TAPP2. TAPP KI mice show evidence of altered B-cell development, but generate phenotypically normal mature B-cell populations. Total serum immunoglobulin IgM and IgG levels were found to be markedly elevated in TAPP KI mice. B cells purified from TAPP KI mice were hyper-responsive to antigen receptor cross-linking, showing increased proliferation, CD86 expression, and Akt phosphorylation on Ser473 and Thr308. Female TAPP KI mice developed elevated levels of anti-DNA and antinuclear antibodies with age, associated with IgG deposition in kidneys and significant glomerulonephritis pathology. Together our results indicate that interaction of TAPPs with PI(3,4)P2 mediates feedback inhibition impacting on BCR signaling, with functional significance for control of autoreactive B cells.

Anti-CD20 multivalent HPMA copolymer-Fab′ conjugates for the direct induction of apoptosis

A hybrid biomimetic system comprising high-molecular-weight, linear copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) grafted with multiple Fab′ fragments of anti-CD20 monoclonal antibody (mAb) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by attachment of Fab′ fragments via thioether bonds. Exposure of human non-Hodgkin’s lymphoma (NHL) Raji B cells to the multivalent conjugates resulted in crosslinking of CD20 receptors and commencement of apoptosis. Five conjugates with varying molecular weight and valence (amount of Fab′ per polymer chain) were synthesized. One of the copolymers contained enzyme degradable peptide sequences (GFLG) in the backbone. The multivalency led to higher avidity and apoptosis induction compared to unconjugated whole mAb. Time-dependent studies showed that the cytotoxicity of conjugates exhibited a slower onset at shorter exposure times than mAb hyper-crosslinked with a secondary Ab; however, at longer time intervals the HPMA copolymer conjugates achieved significantly higher biological efficacies. In addition, study of the relationship between the structure of conjugates and Raji B cell apoptosis revealed that both valency and polymer molecular weight influenced biological activities, while insertion of peptide sequences into the backbone was not a factor in vitro.

Innate immune responses to rhinovirus are reduced by the high-affinity IgE receptor in allergic asthmatic children

Children with allergic asthma have more frequent and severe human rhinovirus (HRV)-induced wheezing and asthma exacerbations through unclear mechanisms. We sought to determine whether increased high-affinity IgE receptor (FcεRI) expression and cross-linking impairs innate immune responses to HRV, particularly in allergic asthmatic children. PBMCs were obtained from 44 children, and surface expression of FcεRI on plasmacytoid dendritic cells (pDCs), myeloid dendritic cells, monocytes, and basophils was assessed by using flow cytometry. Cells were also incubated with rabbit anti-human IgE to cross-link FcεRI, followed by stimulation with HRV-16, and IFN-α and IFN-λ1 production was measured by Luminex. The relationships among FcεRI expression and cross-linking, HRV-induced IFN-α and IFN-λ1 production, and childhood allergy and asthma were subsequently analyzed. FcεRIα expression on pDCs was inversely associated with HRV-induced IFN-α and IFN-λ1 production. Cross-linking FcεRI before HRV stimulation further reduced PBMC IFN-α (47% relative reduction; 95% CI, 32% to 62%; P< .0001) and IFN-λ1 (81% relative reduction; 95% CI, 69% to 93%; P< .0001) secretion. Allergic asthmatic children had higher surface expression of FcεRIα on pDCs and myeloid dendritic cells when compared with that seen in nonallergic nonasthmatic children. Furthermore, after FcεRI cross-linking, allergic asthmatic children had significantly lower HRV-induced IFN responses than allergic nonasthmatic children (IFN-α, P= .004; IFN-λ1, P= .02) and nonallergic nonasthmatic children (IFN-α, P= .002; IFN-λ1, P= .01). Allergic asthmatic children have impaired innate immune responses to HRV that correlate with increased FcεRI expression on pDCs and are reduced by FcεRI cross-linking. These effects likely increase susceptibility to HRV-induced wheezing and asthma exacerbations.

Mechanistic Insight into Pertussis Toxin and Lectin Signaling Using T Cells Engineered To Express a CD8α/CD3ζ Chimeric Receptor

Mammalian cell-surface receptors typically display N- or O-linked glycans added post-translationally. Plant lectins such as phytohemagluttinin (PHA) can activate the T cell receptor (TCR) and other cell-surface receptors by binding to glycans and initiating receptor cross-linking. Pathogenic microorganisms such as Bordetella pertussis also express proteins with lectin-like activities. Similar to plant lectins, pertussis toxin (PTx) can activate the TCR and bind to a variety of glycans. However, whether the lectin-like activity of PTx is responsible for its ability to activate TCR signaling has not been formally proven. Here we examined the ability of PTx and a panel of lectins to activate the TCR or a CD8α/CD3ζ chimeric receptor (termed CD8ζ). We demonstrate that CD8ζ rescues PTx-induced signaling events lacking in TCR null cells. This result indicates that CD8ζ can substitute for TCR and supports the hypothesis that PTxB (functioning as a lectin) stimulates signaling via receptor cross-linking rather than by binding to a specific epitope on the TCR. Moreover, PTx is able to activate signaling by binding either N-linked or O-linked glycan-modified receptors as the TCR displays N-linked glycans while CD8ζ displays O-linked glycans. Finally, studies with a diverse panel of lectins indicate that the signaling activity of the lectins does not always correlate with the biochemical reports of ligand preferences. Comparison of lectin signaling through TCR or CD8ζ allows us to better define the structural and functional properties of lectin-glycan interactions using a biologically based signaling readout.

Disruption of Fnip1 Reveals a Metabolic Checkpoint Controlling B Lymphocyte Development

The coordination of nutrient and energy availability with cell growth and division is essential for proper immune cell development and function. By usinga chemical mutagenesis strategy in mice, we identified a pedigree that has a complete block in B cell development at the pre-B cell stage resulting from a deletion in the Fnip1 gene. Enforced expression of an immunoglobulin transgene failed to rescue B cell development. Whereas essential pre-B cell signaling molecules were activated normally in Fnip1-null pre-B cells, the metabolic regulators AMPK and mTOR were dysregulated, resulting in excessive cell growth and enhanced sensitivity to apoptosis in response to metabolic stress (pre-B cell receptor crosslinking, oncogene activation). These results indicate that Folliculin-interacting protein 1 (Fnip1) is vital for B cell development and metabolic homeostasis and reveal a metabolic checkpoint that may ensure that pre-B cells have sufficient metabolic capacity to support division, while limiting lymphomagenesis caused by deregulated growth.

Developmentally regulated expression of MEF2C limits the response to BCR engagement in transitional B cells

Transitional and naïve mature peripheral B cells respond very differently to B-cell receptor (BCR) cross-linking. While transitional B cells undergo apoptosis upon BCR engagement, mature B cells survive and proliferate. This differential response correlates with the capacity of mature, but not transitional B cells to transcribe genes that promote cell survival and proliferation, including those encoding c-Myc and the Bcl-2 family members Bcl-xL and A1. We recently demonstrated that transitional B cells fail to assemble transcriptional machinery at the promoter region of these target genes despite equivalent cytoplasmic signaling and nuclear translocation of key transcription factors including NF-κB and nuclear factor of activated T cells (NFAT). The transcription factor myocyte enhancer factor-2C (MEF2C) is regulated by both calcineurin and mitogen-activated protein kinase signaling pathways, and is essential for proliferation and survival downstream of BCR engagement in mature B cells. In this work, we demonstrate that transitional B cells have intrinsically low levels of MEF2C protein and DNA-binding activity, and that this developmental difference in MEF2C expression is functionally significant. Forced expression of MEF2C in transitional B cells promoted cell survival, proliferation, and upregulation of pro-survival genes. Thus, low MEF2C expression limits transitional B-cell responsiveness to BCR engagement before these cells reach maturity.