bluuebunny/crossref_metadata_2025 huggingface dataset:
A Fatal Cytokine-Induced Systemic Inflammatory Response Reveals a Critical Role for NK Cells
Authors: Carson William E. , Yu Haixin , Dierksheide Julie , Pfeffer Klaus , Bouchard Page , Clark Reed , Durbin Joan , Baldwin Albert S. , Peschon Jacques , Johnson Philip R. , Ku George , Baumann Heinz , Caligiuri Michael A.
Published: January 2023

The mechanism of cytokine-induced shock remains poorly understood. The combination of IL-2 and IL-12 has synergistic antitumor activity in vivo, yet has been associated with significant toxicity. We examined the effects of IL-2 plus IL-12 in a murine model and found that the daily, simultaneous administration of IL-2 and IL-12 resulted in shock and 100% mortality within 4 to 12 days depending on the strain employed. Mice treated with IL-2 plus IL-12 exhibited NK cell apoptosis, pulmonary edema, degenerative lesions of the gastrointestinal tract, and elevated serum levels of proinflammatory cytokines and acute phase reactants. The actions of TNF-α, IFN-γ, macrophage-inflammatory protein-1α, IL-1, IL-1-converting enzyme, Fas, perforin, inducible nitric oxide synthase, and STAT1 did not contribute to the observed toxicity, nor did B or T cells. However, toxicity and death from treatment with IL-2 plus IL-12 could be completely abrogated by elimination of NK cells. These results suggest that the fatal systemic inflammatory response induced by this cytokine treatment is critically dependent upon NK cells, but does not appear to be mediated by the known effector molecules of this cellular compartment. These data may provide insight into the pathogenesis of cytokine-induced shock in humans.
4-1BBL Cooperates with B7-1 and B7-2 in Converting a B Cell Lymphoma Cell Line into a Long-Lasting Antitumor Vaccine
Authors: Guinn Barbara-ann , DeBenedette Mark A. , Watts Tania H. , Berinstein Neil L.
Published: January 2023

A20 is a B cell lymphoma that constitutively expresses the costimulatory molecule B7-2 yet grows readily as a tumor in syngeneic BALB/c mice. We have compared the tumorigenicity of A20 variants expressing either B7-1 (A20/B7-1) or B7-2 (A20/B7-2) with an A20 variant expressing B7-1 and B7-2 with 4-1BBL (A20/4-1BBL), a costimulatory member of the TNF family. Mice injected with tumors expressing the vector backbone (A20/CMV) or B7-1 developed tumors within 25 days of s.c. injection. In contrast, mice injected with A20/4-1BBL were tumor free for the 150-day follow-up period, while 25% of mice injected with A20/B7-2 developed tumors. Tumorigenicity experiments using nude mice indicated the requirement for T cells for variant rejection. Almost all mice that resisted the initial tumor challenge were resistant to further challenge with the parental tumor. Splenocytes from these mice showed high CTL lytic activity against the parental tumor, A20, as well as the syngeneic BALB/c lymphoma K46J, but showed background levels of lytic activity against the congenic SCID thymoma line ST-D2 or the allogeneic EL4 thymoma. In vitro blocking experiments with anti-B7-1 plus anti-B7-2 and/or soluble 4-1BB receptor showed B7-1, B7-2, and 4-1BBL all contributed to the CTL activity. Thus, the data show that neither B7-1 or B7-2 alone can confer full immunogenicity to the A20 lymphoma but that the addition of 4-1BBL results in a tumor that is highly immunogenic and can confer long-lasting protection against challenge with parental tumor in vivo.
Induction of Cyclooxygenase-2 on Activated T Lymphocytes: Regulation of T Cell Activation by Cyclooxygenase-2 Inhibitors
Authors: Iñiguez Miguel A. , Punzón Carmen , Fresno Manuel
Published: January 2023

Cyclooxygenase (COX), known to exist in two isoforms, COX-1 and COX-2, is a key enzyme in prostaglandin synthesis and the target for most nonsteroidal anti-inflammatory drugs. In this study, we show that human T lymphocytes express the COX-2 isoenzyme. COX-2 mRNA and protein were induced in both Jurkat and purified T cells stimulated by TCR/CD3 or PMA activation. COX-2 mRNA was induced very early after activation and superinduced by protein synthesis inhibitors, whereas it was inhibited by the immunosuppressive drug cyclosporin A, identifying it as an early T cell activation gene. Interestingly, treatment with COX-2-specific inhibitors such as NS398 or Celecoxib severely diminished early and late events of T cell activation, including CD25 and CD71 cell surface expression, IL-2, TNF-α, and IFN-γ production and cell proliferation, but not the expression of CD69, an immediate early gene. COX-2 inhibitors also abolished induced transcription of reporter genes driven by IL-2 and TNF-α promoters. Moreover, induced transcription from NF-κB- and NF-AT-dependent enhancers was also inhibited. These results may have important implications in anti-inflammatory therapy and open a new field on COX-2-selective nonsteroidal anti-inflammatory drugs as modulators of the immune activation.
Immunogenicity of Apoptotic Cells In Vivo: Role of Antigen Load, Antigen-Presenting Cells, and Cytokines
Authors: Ronchetti Anna , Rovere Patrizia , Iezzi Giandomenica , Galati Giacomo , Heltai Silvia , Protti Maria Pia , Garancini Maria Paola , Manfredi Angelo A. , Rugarli Claudio , Bellone Matteo
Published: January 2023

Apoptosis allows the clearance of unwanted cells from living tissues without causing inflammation. Processing of phagocytosed apoptotic cells yields Ags that access the cytosol and the MHC class I pathway of engulfing cells and are recognized by Ag-specific CTL. We show here that injection of apoptotic RMA cells, a syngeneic T cell lymphoma, into C57BL/6 mice results in priming of a functional and long-lasting tumor-specific immune response. Cross-priming of CTLs by apoptotic cells requires CD4+ T cell help. Apoptotic cells, however, are at least 20-fold less immunogenic than nonreplicating live cells. Immunogenicity of apoptotic cells is proportional to the number of cells injected, correlates with the serum concentration of IL-10 and IL-1β cytokines, and is enhanced in IL-10 knockout mice. Moreover, immunization with dendritic cells (DCs), but not macrophages (Mφ), pulsed with apoptotic cells primes tumor-specific CTLs and confers protection against a tumor challenge. Our findings demonstrate that tumor cells undergoing apoptosis are, though scarcely, immunogenic in vivo, outline the different roles of Mφ and DCs in the physiologic clearance of unwanted cells, and have implications in designing immunomodulating vaccines.
Anergic CD8+ T Cells Can Persist and Function In Vivo
Authors: Blish Catherine A. , Dillon Stacey R. , Farr Andrew G. , Fink Pamela J.
Published: January 2023

Using a mouse model system, we demonstrate that anergic CD8+ T cells can persist and retain some functional capabilities in vivo, even after the induction of tolerance. In TCR Vβ5 transgenic mice, mature CD8+Vβ5+ T cells transit through a CD8lowVβ5low deletional intermediate during tolerance induction. CD8low cells are characterized by an activated phenotype, are functionally compromised in vitro, and are slated for deletion in vivo. We now demonstrate that CD8low cells derive from a proliferative compartment, but do not divide in vivo. CD8low cells persist in vivo with a t1/2 of 3–5 days, in contrast to their in vitro t1/2 of 0.5–1 day. During this unexpectedly long in vivo life span, CD8low cells are capable of producing IFN-γ in vivo despite their inability to proliferate or to kill target cells in vitro. CD8low cells also accumulate at sites of inflammation, where they produce IFN-γ. Therefore, rather than withdrawing from the pool of functional CD8+ T cells, anergic CD8low cells retain a potential regulatory role despite losing their capacity to proliferate. The ability of anergic cells to persist and function in vivo adds another level of complexity to the process of tolerance induction in the lymphoid periphery.
Predicting Regional Mutability in Antibody V Genes Based Solely on Di- and Trinucleotide Sequence Composition
Authors: Shapiro Gary S. , Aviszus Katja , Ikle David , Wysocki Lawrence J.
Published: January 2023

Somatic mutations are not distributed randomly throughout Ab V region genes. A sequence-specific target bias is revealed by a defined hierarchy of mutability among di- and trinucleotide sequences located within Ig intronic DNA. Here we report that the di- and trinucleotide mutability preference pattern is shared by mouse intronic JH and Jκ clusters and by human VH genes, suggesting that a common mutation mechanism exists for all Ig V genes of both species. Using di- and trinucleotide target preferences, we performed a comprehensive analysis of human and murine germline V genes to predict regional mutabilities. Heavy chain genes of both species exhibit indistinguishable patterns in which complementarity-determining region 1 (CDR1), CDR2, and framework region 3 (FR3) are predicted to be more mutable than FR1 and FR2. This prediction is borne out by empirical mutation data from nonproductively rearranged human VH genes. Analysis of light chain genes in both species also revealed a common, but unexpected, pattern in which FR2 is predicted to be highly mutable. While our analyses of nonfunctional Ig genes accurately predicts regional mutation preferences in VH genes, observed relative mutability differences between regions are more extreme than expected. This cannot be readily accounted for by nascent mRNA secondary structure or by a supplemental gene conversion mechanism that might favor nucleotide replacements in CDR. Collectively, our data support the concept of a common mutation mechanism for heavy and light chain genes of mice and humans with regional bias that is qualitatively, but not quantitatively, accounted for by short nucleotide sequence composition.
Surfactant Protein D Binds to Mycobacterium tuberculosis Bacilli and Lipoarabinomannan via Carbohydrate-Lectin Interactions Resulting in Reduced Phagocytosis of the Bacteria by Macrophages1
Authors: Ferguson J. Scott , Voelker Dennis R. , McCormack Francis X. , Schlesinger Larry S.
Published: January 2023

Surfactant protein-D (SP-D) is a collectin produced in the distal lung airspaces that is believed to play an important role in innate pulmonary immunity. Naive immunologic responses to Mycobacterium tuberculosis (M.tb) are especially important in the lung, since entry of this inhaled pathogen into the alveolar macrophage is a pivotal event in disease pathogenesis. Here we investigated SP-D binding to M.tb and the effect of this binding on the adherence of M.tb to human macrophages. These studies demonstrate specific binding of SP-D to M.tb that is saturable, calcium dependent, and carbohydrate inhibitable. In addition to purified SP-D, SP-D in bronchoalveolar lavage fluids from healthy donors and patients with alveolar proteinosis also binds to M.tb. Incubation of M.tb with SP-D results in agglutination of the bacteria. In contrast to its binding to M.tb, SP-D binds minimally to the avirulent Mycobacterium smegmatis. SP-D binds predominantly to lipoarabinomannan from the virulent Erdman strain of M.tb, but not the lipoarabinomannan from M. smegmatis. The binding of SP-D to Erdman lipoarabinomannan is mediated by the terminal mannosyl oligosaccharides of this lipoglycan. Incubation of M.tb with subagglutinating concentrations of SP-D leads to reduced adherence of the bacteria to macrophages (62.7% of control adherence ± 3.3% SEM, n = 8), whereas incubation of bacteria with surfactant protein A leads to significantly increased adherence to monocyte-derived macrophages. These data provide evidence for specific binding of SP-D to M. tuberculosis and indicate that SP-D and surfactant protein A serve different roles in the innate host response to this pathogen in the lung.
P-Selectin Binding Promotes the Adhesion of Monocytes to VCAM-1 Under Flow Conditions
Authors: Yago Tadayuki , Tsukuda Mamoru , Minami Mutsuhiko
Published: January 2023

This study examined the adhesive interaction of peripheral blood monocytes with VCAM-1 and analyzed the effect of P-selectin binding to monocytes on the adhesive interaction with VCAM-1 under flow conditions. P-selectin glycoprotein ligand-1 is expressed on most monocytes. Furthermore, most monocytes bind soluble P-selectin derived from platelets. P-selectin binding to monocytes did not alter the amount of expression of α4 integrin on monocytes. However, the mean channel fluorescence value for binding Cy2-conjugated soluble VCAM-1 to P-selectin-bound monocytes was slightly more than that for binding Cy2-conjugated soluble VCAM-1 to untreated monocytes. Under flow conditions, the number of P-selectin-bound monocytes bound to VCAM-1 was much higher than that of untreated monocytes bound to VCAM-1. These bindings were abolished by pretreatment of untreated monocytes and P-selectin-bound monocytes with anti-VCAM-1 mAb or anti-α4 integrin mAb. Furthermore, P-selectin binding to monocytes increased shear resistance and thus increased the adhesive strength of monocytes to VCAM-1. These findings indicate that P-selectin binding to monocytes enhances the adhesive interaction of monocytes with VCAM-1. It is suggested that P-selectin glycoprotein ligand-1/P-selectin interaction and α4 integrin/VCAM-1 interaction can act sequentially in the adhesion cascade that regulates monocyte trafficking to inflammatory and atherosclerotic lesion.
Induction of Phosphorylation and Intracellular Association of CC Chemokine Receptor 5 and Focal Adhesion Kinase in Primary Human CD4+ T Cells by Macrophage-Tropic HIV Envelope
Authors: Cicala Claudia , Arthos James , Ruiz Margaret , Vaccarezza Mauro , Rubbert Andrea , Riva Agostino , Wildt Kathryn , Cohen Oren , Fauci Anthony S.
Published: January 2023

Binding of HIV-1 envelope glycoproteins to the surface of a CD4+ cell transduces intracellular signals through the primary envelope receptor, CD4, and/or the envelope coreceptor, a seven-transmembrane chemokine receptor. Macrophage-tropic strains of HIV-1 preferentially use CCR5 as an entry coreceptor, whereas T cell-tropic strains use CXC chemokine receptor-4 for entry. Intracellular signals transduced by HIV-1 envelope may have immunopathogenic consequences, including anergy, syncytium formation, apoptosis, and inappropriate cell trafficking. We demonstrate here that a recombinant envelope protein derived from an M-tropic isolate of HIV-1 can transduce CD4-dependent as well as CCR5-dependent intracellular signals in primary human CD4+ T cells. Novel HIV-induced intracellular signals that were identified include tyrosine phosphorylation of focal adhesion kinase (FAK) and CCR5, which are involved in cell adhesion and chemotaxis, respectively. HIV envelope-induced cellular association of FAK and CCR5 was also demonstrated, suggesting that ligation of CD4 and CCR5 leads to the formation of an activation complex composed of FAK and CCR5. Activation of this signaling pathway by HIV-1 envelope may be an important pathogenic mechanism of dysregulated cellular activation and trafficking during HIV infection.
IL-5 Increases Expression of 5-Lipoxygenase-Activating Protein and Translocates 5-Lipoxygenase to the Nucleus in Human Blood Eosinophils
Authors: Cowburn Andrew S. , Holgate Stephen T. , Sampson Anthony P.
Published: January 2023

Cysteinyl-leukotrienes are potent bronchoconstrictor mediators synthesized by the 5-lipoxygenase (5-LO) pathway. Eosinophilopoietic cytokines such as IL-5 enhance cysteinyl-leukotriene synthesis in eosinophils in vitro, mimicking changes in eosinophils from asthmatic patients, but the mechanism is unknown. We hypothesized that IL-5 induces the expression of 5-LO and/or its activating protein FLAP in eosinophils, and that this might be modulated by anti-inflammatory corticosteroids. Compared with control cultures, IL-5 increased the proportion of normal blood eosinophils immunostaining for FLAP (65 ± 4 vs 34 ± 4%; p < 0.0001), enhanced immunoblot levels of FLAP by 51 ± 14% (p = 0.03), and quadrupled ionophore-stimulated leukotriene C4 synthesis from 5.7 to 20.8 ng/106 cells (p < 0.02). IL-5 effects persisted for 24 h and were abolished by cycloheximide and actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (p < 0.0001). Neither IL-5 nor dexamethasone altered 5-LO expression, but IL-5 significantly increased 5-LO immunofluorescence localizing to eosinophil nuclei. Compared with normal subjects, allergic asthmatic patients had a greater proportion of circulating FLAP+ eosinophils (46 ± 6 vs 27 ± 3%; p < 0.03) and a smaller IL-5-induced increase in FLAP immunoreactivity (p < 0.05). Thus, IL-5 increases FLAP expression and translocates 5-LO to the nucleus in normal blood eosinophils in vitro. This is associated with an enhanced capacity for cysteinyl-leukotriene synthesis and mimics in vivo increases in FLAP expression in eosinophils from allergic asthmatics.
Cutting Edge: SLP-76 Cooperativity with FYB/FYN-T in the Up-Regulation of TCR-Driven IL-2 Transcription Requires SLP-76 Binding to FYB at Tyr595 and Tyr651
Authors: Geng Liping , Raab Monika , Rudd Christopher E.
Published: January 2023

SLP-76 (Src homology (SH) 2-domain-containing leukocyte protein of 76 kDa) and FYB/SLAP (FYN-T-binding protein/SLP-76-associated protein) are two hemopoietic cell-specific adaptor proteins downstream of TCR-activated protein tyrosine kinases. SLP-76 has been implicated as an essential component in T cell signaling. FYB is selectively phosphorylated by FYN-T, providing a template for the recruitment of FYN-T and SLP-76 SH2 domains. Coexpression of FYN-T, FYB, and SLP-76 can synergistically up-regulate IL-2 production in T cells upon TCR ligation. In this report, we show that two tyrosines, Tyr595 and Tyr651, of FYB are major sites of phosphorylation by FYN-T and mediate binding to SLP-76 in Jurkat T cells. Furthermore, the synergistic up-regulation of IL-2 promoter activity in the FYN-T-FYB-SLP-76 pathway is contingent upon the interaction between FYB and SLP-76, but not the interaction between FYB and FYN-T. These observations define a pathway by which SLP-76 interacts with downstream components in the up-regulation of T cell cytokine production.
Sustained Activation of the Raf-MEK-ERK Pathway Elicits Cytokine Unresponsiveness in T Cells
Authors: Chen Dan , Heath Victoria , O’Garra Anne , Johnston James , McMahon Martin
Published: January 2023

Activation of T cells via the TCR and other costimulatory receptors triggers a number of signaling cascades. Among them, the Ras-activated Raf-mitogen-activated protein/extracellular signal-related kinase (ERK) kinase (MEK)-ERK signaling cascade has been demonstrated to be crucial for both T cell development and activation. It has previously been demonstrated that high doses of Ag or anti-CD3 mAb are able to induce in T cells a nonresponsive state to subsequent treatment with cytokines such as IL-2. The precise biochemical mechanisms underlying this effect are not fully characterized. In this study, we demonstrate that cytokine nonresponsiveness is accompanied by the induction of the cyclin-dependent kinase inhibitor p21Cip1 that is mediated, at least in part, by the activation of the Raf-MEK-ERK pathway. Furthermore, we demonstrate that selective activation of the Raf-MEK-ERK signaling pathway in T cells is sufficient to induce cytokine nonresponsiveness in both a T cell clone and naive primary T cells. In this case, nonresponsiveness is accompanied by the induction of p21Cip1 and the prevention of p27Kip1 down-regulation, leading to inhibition of cyclin E/cyclin-dependent kinase 2 activity. These data suggest that anti-CD3 mAb-induced cytokine nonresponsiveness may be a consequence of hyperactivation of the Raf-MEK-ERK pathway, leading to alterations in the expression of key cell cycle regulators. These observations may provide a novel insight into the mechanisms of induction of peripheral tolerance.
Protein Kinase Cθ Cooperates with Calcineurin to Induce Fas Ligand Expression During Activation-Induced T Cell Death
Authors: Villalba Martin , Kasibhatla Shailaja , Genestier Laurent , Mahboubi Artin , Green Douglas R. , Altman Amnon
Published: January 2023

Activation-induced cell death is mediated by the TCR-induced expression of the Fas ligand (FasL) on the surface of T cells, followed by binding to its receptor Fas. FasL expression is induced by stimulating T cells with a combination of phorbol ester and Ca2+ ionophore, implicating a role for protein kinase C (PKC) in this process. However, the precise mechanisms that regulate FasL expression, including the contribution of distinct T cell-expressed PKC isoforms, are poorly understood. Herein, we report that PKCθ, a Ca2+-independent PKC isoform that we have previously isolated as a PKC enzyme selectively expressed in T cells, plays an important role in these processes. A constitutively active PKCθ mutant preferentially induced FasL expression and activated the corresponding gene promoter; conversely, a dominant-negative PKCθ mutant blocked FasL expression induced by anti-CD3 or PMA plus ionomycin stimulation. Furthermore, PKCθ synergized with calcineurin to provide a potent stimulus for FasL promoter activation. Full activation of the promoter required its binding sites for the transcription factors NF-AT, AP-1, and NF-κB. The biological significance of these findings is implicated by the finding that rottlerin, a selective PKCθ inhibitor, blocked FasL induction by anti-CD3 or PMA plus ionomycin stimulation and, consequently, protected human Jurkat T cells and the mouse T cell hybridoma A1.1 from activation-induced cell death.
Augmentation of an Antitumor CTL Response In Vivo by Inhibition of Suppressor Macrophage Nitric Oxide
Authors: Medot-Pirenne Marianne , Heilman Michelle J. , Saxena Malinee , McDermott Patricia E. , Mills Charles D.
Published: January 2023

Evidence is provided that inhibition of macrophage NO production can augment in vivo CTL responses. Specifically, administration of NG-monomethyl-l-arginine (NGMMA) via osmotic pumps increases the tumor-specific CTL response against the P815 mastocytoma in the peritoneal cavity of preimmunized mice. Both the magnitude and duration of the CTL response were increased. That the augmented CTL response resulted from inhibition of the NO synthase pathway is supported by the finding that macrophage NO production from NGMMA-treated mice was reduced. Also, in vitro inhibition of NO production by peritoneal exudate cells from P815 tumor-challenged mice augmented the secondary CTL response observed. Cell proliferation was augmented by NGMMA in these cultures, suggesting that macrophage NO may suppress CTL by inhibiting clonal expansion. NO-mediated inhibition was observed in vivo in this experimental system, even though the CTL response is not suppressed, in that tumor rejection occurs. Therefore, the present results are consistent with the conclusion that macrophage NO-mediated inhibition of the CTL response is a side effect of activating macrophages rather than resulting from the action of a distinct subset of what have long been termed suppressor macrophages. Most important, the results indicate that NO-mediated suppressor macrophage activity can be an important CTL immunoregulatory element in vivo.
Proliferation of Bone Marrow Pro-B Cells Is Dependent on Stimulation by the Pituitary/Thyroid Axis
Authors: Foster Melanie P. , Montecino-Rodriguez Encarnacion , Dorshkind Kenneth
Published: January 2023

The frequency and absolute number of pro-B, pre-B, and B cells in the bone marrow of the hypothyroid strain of mice are significantly reduced compared with those of their normal littermates. To investigate why this is the case, various B cell developmental processes were examined in the thyroid hormone-deficient mice. These studies revealed that the frequency of pro-B cells in the S-G2/M phase of the cell cycle was significantly reduced in hypothyroid mice. That thyroid hormone deficiency was responsible for this proliferation defect was established by demonstrating that treatment of hypothyroid mice with thyroxine resulted in a specific increase in the frequency and total number of cycling pro-B cells. The latter effect was paralleled by increases in the frequency and number of bone marrow B lineage cells. Additional in vitro experiments revealed that at least some thyroid hormone effects were directly mediated on the bone marrow. Taken together, these data demonstrate that thyroid hormones are required for normal B cell production in the bone marrow through regulation of pro-B cell proliferation and establish a role for the pituitary/thyroid axis in B cell development.
Requirement for Stat5 in Thymic Stromal Lymphopoietin-Mediated Signal Transduction
Authors: Isaksen Deborah E. , Baumann Heinz , Trobridge Patty A. , Farr Andrew G. , Levin Steven D. , Ziegler Steven F.
Published: January 2023

Thymic stromal lymphopoietin (TSLP) is a newly identified cytokine that uniquely promotes B lymphopoiesis to the B220+/IgM+ immature B cell stage. In addition, TSLP shares many biological properties with the related cytokine IL-7. This can be explained by the finding that the receptor complexes for TSLP and IL-7 both contain the IL-7R α-chain; IL-7Rα is paired with the common γ-chain (γc) in the IL-7 receptor complex and the unique TSLP-R chain in the TSLP receptor complex. Although TSLP and IL-7 both induce tyrosine phosphorylation of the transcription factor Stat5, only IL-7-mediated signal transduction could be associated with activation of Janus family kinases (Jaks). Because Stat5 phosphorylation following cytokine stimulation is generally mediated by Jaks, the lack of Jak activation after TSLP treatment suggested the possibility that tyrosine-phosphorylated Stat5 may be nonfunctional. Herein, we demonstrate that TSLP induces a functional Stat5 transcription factor in that TSLP stimulation results in Stat5-DNA complex formation and transcription of the Stat5-responsive gene CIS. We also show that the TSLP receptor complex is functionally reconstituted using TSLP-R and IL-7Rα and that TSLP-mediated signal transduction requires Stat5. Moreover, TSLP-mediated signaling is inhibited by suppressor of cytokine signaling (SOCS)-1 and a kinase-deficient version of Tec but not by kinase-deficient forms of Jak1 and Jak2.
High Affinity IgG Receptor Activation of Src Family Kinases Is Required for Modulation of the Shc-Grb2-Sos Complex and the Downstream Activation of the Nicotinamide Adenine Dinucleotide Phosphate (Reduced) Oxidase
Authors: Park Rae-Kil , Erdreich-Epstein Anat , Liu Ming , Izadi Kayvon D. , Durden Donald L.
Published: January 2023

We used the U937 cell line to examine the modulation of adaptor protein interactions (Shc, Grb2, and Cbl) after high affinity IgG receptor (FcγRI) cross-linking, leading to the formation of the Grb2-Sos complex, the activation of Ras, and the regulation of the respiratory burst. Cross-linking of FcγRI induced the conversion of GDP-Ras to GTP-Ras reaching a maximum 5 min after stimulation. Concomitant with Ras activation, Sos underwent an electrophoretic mobility shift and the Sos-Grb2 association was increased (6-fold). The Grb2-Sos complex was present only in the membrane fraction and was augmented after FcγRI stimulation. Tyrosine-phosphorylated Shc, mainly the p52 isoform, was observed to transiently onload to the membrane Grb2-Sos complex on FcγRI stimulation. Cross-linking of FcγRI induces the tyrosine phosphorylation of Cbl, which forms a complex with Grb2 and Shc via the Cbl C terminus. Kinetic experiments confirm that Cbl-Grb2 is relatively stable, whereas Grb2-Sos, Grb2-Shc, and Cbl-Shc interactions are highly inducible. The Src family tyrosine kinase inhibitor, PP1, was shown to completely inhibit Shc tyrosine phosphorylation, the Shc-Grb2 interaction, and the FcγR-induced respiratory burst. Our results provide the first evidence that the upstream activation of Src kinases is required for the modulation of the Shc-Grb2 interaction and the myeloid NADPH oxidase response.
Recruitment of CREB-Binding Protein by PU.1, IFN-Regulatory Factor-1, and the IFN Consensus Sequence-Binding Protein Is Necessary for IFN-γ-Induced p67phoxand gp91phoxExpression
Authors: Eklund Elizabeth A. , Kakar Renu
Published: January 2023

Activation of the phagocyte respiratory burst oxidase requires interaction between the oxidase components p47phox, p67phox, p22phox, and gp91phox. IFN-γ induces transcription of the genes encoding p67phox (the NCF2 gene) and gp91phox (the CYBB gene) during monocyte differentiation, and also in mature monocytes. In these studies, we identify an NCF2 cis element, necessary for IFN-γ-induced p67phox expression, and determine that this element is activated by cooperation between the transcription factors PU.1, IFN regulatory factor 1 (IRF1), and the IFN consensus-binding protein (ICSBP). Previously, we identified a CYBB cis element, necessary for IFN-γ-induced gp91phox expression, and also activated by this transcription factor combination. In these investigations, we determine that recruitment of a coactivator protein, CBP (the CREBbinding protein), to the CYBB or NCF2 promoter is the molecular mechanism of transcriptional activation by PU.1, IRF1, and ICSBP. Also, we determine that the multiprotein interaction of CBP with PU.1, IRF1, and ICSBP requires either the CYBB- or NCF2--binding site. Because IFN-γ induces simultaneous expression of p67phox and gp91phox, these investigations identify a molecular event that coordinates oxidase gene transcription during the inflammatory response. Also, these investigations identify CBP recruitment by cooperation between PU.1, IRF1, and ICSBP as a novel molecular mechanism for IFN-γ-induced activation of myeloid genes that are involved in the system of host defense.
Lipoteichoic Acid inhibits Lipopolysaccharide-Induced Adhesion Molecule Expression and IL-8 Release in Human Lung Microvascular Endothelial Cells
Authors: Blease Kate , Chen Yan , Hellewell Paul G. , Burke-Gaffney Anne
Published: January 2023

Cell adhesion molecule expression (CAM) and IL-8 release in lung microvascular endothelium facilitate neutrophil accumulation in the lung. This study investigated the effects of lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, alone and with LPS or TNF-α, on CAM expression and IL-8 release in human lung microvascular endothelial cells (HLMVEC). The concentration-dependent effects of Staphylococcus aureus (S. aureus) LTA (0.3–30 μg/ml) on ICAM-1 and E-selectin expression and IL-8 release were bell shaped. Streptococcus pyogenes (S. pyogenes) LTA had no effect on CAM expression, but caused a concentration-dependent increase in IL-8 release. S. aureus and S. pyogenes LTA (30 μg/ml) abolished LPS-induced CAM expression, and S. aureus LTA reduced LPS-induced IL-8 release. In contrast, the effects of S. aureus LTA with TNF-α on CAM expression and IL-8 release were additive. Inhibitory effects of LTA were not due to decreased HLMVEC viability, as assessed by ethidium homodimer-1 uptake. Changes in neutrophil adhesion to HLMVEC paralleled changes in CAM expression. Using RT-PCR to assess mRNA levels, S. aureus LTA (3 μg/ml) caused a protein synthesis-dependent reduction (75%) in LPS-induced IL-8 mRNA and decreased the IL-8 mRNA half-life from >6 h with LPS to ∼2 h. These results suggest that mechanisms exist to prevent excessive endothelial cell activation in the presence of high concentrations of bacterial products. However, inhibition of HLMVEC CAM expression and IL-8 release ultimately may contribute to decreased neutrophil accumulation, persistence of bacteria in the lung, and increased severity of infection.
Peptides of Human Thyroglobulin Reactive with Sera of Patients with Autoimmune Thyroid Disease
Authors: Saboori Ali M. , Rose Noel R. , Yuhasz Stacieann C. , Amzel L. Mario , Burek C. Lynne
Published: January 2023

Autoantibodies to thyroglobulin (Tg) are a prominent feature of the two autoimmune thyroid diseases, chronic lymphocytic (Hashimoto’s) thyroiditis and Graves’ disease. Similar autoantibodies are found in the serum of many normal individuals without evidence of thyroid disease. Previous studies have indicated that patients with autoimmune thyroid disease recognize epitopes of Tg which are not usually recognized by normal individuals. The goal of this investigation was to identify peptide fragments of Tg bearing these disease-associated epitopes. For this purpose, we utilized a panel of mAbs that bind to different epitopes of the Tg molecule. One of these mAbs (137C1) reacted with an epitope that was also recognized by the sera of patients with autoimmune thyroiditis. In the present study, we show that two peptides (15 and 23 kDa) that reacted with mAb 137C1 are located in different parts of the Tg molecule. Each peptide inhibited the binding of mAb 137C1 to the other peptide and to the intact Tg, indicating that the same epitope was represented on the two peptides. Loops and helices of the secondary structure of the two peptides might be involved in the conformational epitope recognized by mAb 137C1. A striking finding of this study is that two apparently unrelated fragments of the Tg molecule bind to the same mAb. These findings may have important ramifications with regard to epitope spread and the progression of the autoimmune response to disease.
Identification of an IL-10-Producing HLA-DR-Negative Monocyte Subset in the Malignant Ascites of Patients with Ovarian Carcinoma That Inhibits Cytokine Protein Expression and Proliferation of Autologous T Cells
Authors: Loercher Amy E. , Nash Micheal A. , Kavanagh John J. , Platsoucas Chris D. , Freedman Ralph S.
Published: January 2023

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites from patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-β2, but not IL-12, IL-1α, or TNF-α, and they expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a, CD11b, or CD25 cell surface Ags. Since this subset of monocytes could affect the modulation of tumor immune responses in vivo, studies were undertaken to determine their effect on the activation and proliferation of autologous T cells from the peritoneal cavity of patients with ovarian carcinoma. Expression of cytokine-specific transcripts in T cells was determined by RT-PCR. Transcripts for the following cytokines were detected in patient specimens that also contained the IL-10-producing monocytes IL-2 (12 of 17 specimens), GM-CSF (9 of 17 specimens), IFN-γ (6 of 17 specimens), and TNF-α (4 of 17 specimens). Cytokine production by T cells was determined by intracellular flow cytometry and by ELISA. IL-2 and IFN-γ proteins, unlike their transcripts, were detected only in specimens that lacked IL-10-producing monocytes. IL-10-producing monocytes cocultured with autologous T cells inhibited the proliferation of the T cells in response to PHA. However, T cells cocultured with PEC from which the IL-10-producing monocytes had been removed did not inhibit T cell proliferation. Moreover, the inhibition of T cell proliferation by IL-10-producing monocytes could be reversed by adding neutralizing Abs to both IL-10R and TGF-β2. These results suggest that this subset of monocytes may modulate immune responses by inhibiting T cell proliferation and cytokine protein production.
Essential Role for Both CD80 and CD86 Costimulation, But Not CD40 Interactions, in Allergen-Induced Th2 Cytokine Production from Asthmatic Bronchial Tissue: Role for αβ, But Not γδ, T Cells
Authors: Jaffar Zeina H. , Stanciu Luminita , Pandit Anita , Lordan James , Holgate Stephen T. , Roberts Kevan
Published: January 2023

CD80 and CD86 interact with CD28 and deliver costimulatory signals required for T cell activation. We demonstrate that ex vivo allergen stimulation of bronchial biopsy tissue from mild atopic asthmatic, but not atopic nonasthmatic, subjects induced production of IL-5, IL-4, and IL-13. Explants from both study groups did not produce IFN-γ, but secreted the chemokine RANTES without any overt stimulation. In addition to allergen, stimulation of asthmatic explants with mAbs to CD3 and TCR-αβ but not TCR-γδ induced IL-5 secretion. Allergen-induced IL-5 and IL-13 production by the asthmatic tissue was inhibited by anti-CD80 and, to a lesser extent, by anti-CD86 mAbs. In contrast, the production of these cytokines by PBMCs was not affected by mAbs to CD80, was inhibited by anti-CD86, and was strongly attenuated in the presence of both Abs. FACS analysis revealed that stimulated asthmatic bronchial tissue was comprised of CD4+ T cells that expressed surface CD28 (75.3%) but little CTLA-4 (4.0%). Neutralizing mAbs to CD40 ligand had no effect on the cytokine levels produced by asthmatic tissue or PBMCs. Collectively, these findings suggest that allergen-specific αβ T cells are resident in asthmatic bronchial tissue and demonstrate that costimulation by both CD80 and CD86 is essential for allergen-induced cytokine production. In contrast, CD86 appears to be the principal costimulatory molecule required in PBMC responses. Attenuation of type 2 αβ T cell responses in the bronchial mucosa by blocking these costimulatory molecules may be of therapeutic potential in asthma.
Eosinophil Chemotactic Chemokines (Eotaxin, Eotaxin-2, RANTES, Monocyte Chemoattractant Protein-3 (MCP-3), and MCP-4), and C-C Chemokine Receptor 3 Expression in Bronchial Biopsies from Atopic and Nonatopic (Intrinsic) Asthmatics
Authors: Ying Sun , Meng Qiu , Zeibecoglou Kyriaki , Robinson Douglas S. , Macfarlane Alison , Humbert Marc , Kay A. Barry
Published: January 2023

Atopic (AA) and nonatopic (NAA) asthma are characterized by chronic inflammation and local tissue eosinophilia. Many C-C chemokines are potent eosinophil chemoattractants and act predominantly via the CCR3. We examined the expression of eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), MCP-4, and CCR3 in the bronchial mucosa from atopic (AA) and nonatopic (intrinsic; NAA) asthmatics and compared our findings with atopic (AC) and nonatopic nonasthmatic controls (NC). Cryostat sections were processed for immunohistochemistry (IHC), in situ hybridization (ISH), and double IHC/ISH. Compared with AC and NC, the numbers of EG2+ cells and the cells expressing mRNA for eotaxin, eotaxin-2, RANTES, MCP-3, MCP-4, and CCR3 were significantly increased in AA and NAA (p < 0.01). Nonsignificant differences in these variants were observed between AA and NAA and between AC and NC. Significant correlations between the cells expressing eotaxin or CCR3 and EG2+ eosinophils in the bronchial tissue were also observed for both AA (p < 0.01) and NAA (p = 0.01). Moreover, in the total asthmatic group (AA + NAA) there was a significant inverse correlation between the expression of eotaxin and that of the histamine PC20 (p < 0.05). Sequential IHC/ISH showed that cytokeratin+ epithelial cells, CD31+ endothelial cells, and CD68+ macrophages were the major sources of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4. There was no significantly different distribution of cells expressing mRNA for these chemokines between atopic and nonatopic asthma. These findings suggest that multiple C-C chemokines, acting at least in part via CCR3, contribute to bronchial eosinophilia in both atopic and nonatopic asthma.
Activation of Regulatory Cells Suppresses Experimental Allergic Encephalomyelitis Via Secretion of IL-10
Authors: Stohlman Stephen A. , Pei Liong , Cua Daniel J. , Li Zhihua , Hinton David R.
Published: January 2023

Suppression of CD4+ Th1 cell-mediated autoimmune disease via immune deviation is an attractive potential therapeutic approach. CD4+ Th2 T cells specific for myelin basic protein, induced by immunization of young adult male SJL mice, suppress or modify the progression of CNS autoimmune disease. This report demonstrates that activation of non-neuroantigen-specific Th2 cells is sufficient to suppress both clinical and histological experimental allergic encephalomyelitis (EAE). Th2 cells were obtained following immunization of male SJL mice with keyhole limpet hemocyanin. Transfer of these cells did not modify EAE, a model of human multiple sclerosis, in the absence of cognate Ag. Disease suppression was obtained following adoptive transfer and subcutaneous immunization. Suppression was not due to the deletion of myelin basic protein-specific T cells, but resulted from the presence of IL-10 as demonstrated by the inhibition of Th2-mediated EAE suppression via passive transfer with either anti-IL-10 or anti-IL-10R mAb. These data demonstrate that peripheral activation of a CD4+ Th2 population specific for an Ag not expressed in the CNS modifies CNS autoimmune disease via IL-10. These data suggest that either peripheral activation or direct administration of IL-10 may be of benefit in treating Th1-mediated autoimmune diseases.
Allergic responses to human rhinoviruses in a mouse model (128.1)
Authors: McLean Gary R , Bartlett Nathan W , Walton Ross P , Johnston Sebastian L
Published: January 2023

Respiratory virus infections are the major cause of acute exacerbations of asthma with human rhinoviruses (RV) accounting for two thirds of infections. Excessive Th2-type responses and deficient anti-viral Th1-type responses generated against RV are associated with exacerbation severity. We recently reported a mouse model of RV infection and showed primary infection was associated with only Th1 responses, however RV infections are frequent throughout life. In a mouse model of repeated RV infection we show the induction of Th2 immune responses, virus-specific IgE and other hallmarks of an allergic response following secondary RV challenge. Both homologous and heterologous RV serotype rechallenges induced eosinophil and lymphocyte infiltration of bronchoalveolar lavage (BAL) and RV-specific serum IgE. The Th2 cytokine IL-4 was detected in BAL 1 day after secondary RV infection, the eosinophil survival/activation factor IL-5 and the chemotactic factor eotaxin were detected in BAL prior to eosinophil cellular infiltration. These results suggest that a Th2-biased immune response generated following repeated RV infection in vivo could contribute to the pathogenesis of asthma exacerbations. With this model it will be possible to dissect out the molecular mechanisms of Th2-biased immune responses to RV and their role in the pathogenesis of RV-induced acute asthma exacerbations.
γδ T cells and NK cells contribute to resistance to polyomavirus–induced tumors (128.15)
Authors: Mishra Rabinarayan , Chen Alex T , Welsh Raymond M , Szomolanyi-Tsuda Eva
Published: January 2023

Infection of adult T cell-deficient (TCR βXδ knockout [KO]) mice with the small DNA tumor virus, polyoma (PyV), induces tumor formation, whereas mice that lack αβ T cells but have γδ T cells remain tumor-free after infection. Moreover, infection of E26 transgenic mice, which lack NK cells and all T cells, leads to an accelerated appearance of virus-induced tumors compared to T cell-deficient mice which have NK cells. These observations suggest that both γδ T cells and NK cells are involved in the resistance to PyV-induced tumors. We found that cell lines established from PyV-induced tumors expressed Rae-1, a ligand for the NKG2D activating receptor expressed on both NK and γδ T cells. Therefore, we hypothesized that Rae-1/ NKG2D interactions were involved in the γδT and NK cell-mediated resistance to PyV-induced tumors. Intraperitoneal injection of the tumor cell lines induced an increase in NK cell number in the peritoneal cavity and also resulted in the activation of NK cells and γδT cells there, manifested by IFNγ and granzyme B production; these changes were not seen in mice intraperitoneally infected with PyV. The Rae-1-expressing PyV tumor cells were also killed by NK cells in in-vitro cytotoxicity assays, and anti-NKG2D antibody treatment prevented this killing. These data strongly suggest that interactions of NKG2D activating receptors on NK and γδ T cells with their ligands expressed on PyV-transformed tumor cells are involved in anti-tumor responses in PyV-infected mice.

Supported by NIH grant CA66644
HLA restricted CTL epitope mapping on Hantaan virus nucleocapsid protein in China (128.7)
Authors: Jin Boquan , Wang Meiliang , Zhu Yong , Wang Jiuping , Fang Liang , Ma Ying , Yang Kun , Yang Angang
Published: January 2023

Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS) in humans. Cytotoxic T lymphocyte (CTL) plays an important role in the clearance of the virus. Only few CTL epitopes specific for HTNV have been identified to date. We analyzed by ex vivo ELISPOT the anti-HTNV CTL responses of peripheral blood mononuclear cells (PBMCs) from patients with HFRS. Seventy 15-mer overlapping peptides spanning the entire HTNV nucleocapsid protein were screened with PBMCs of HFRS patients in Shaanxi province, China. A total of 22 15-mer epitopes were identified, in which 19 were defined as CTL epitopes. Furthermore, 13 minimized 9-mer CTL epitopes were defined on 8 of 19 15-mer peptides, in which three were found to be restricted by various HLA alleles including A24, A11 and B7. IFN-γ ELISPOT analysis showed that memory CTL specific for these epitopes were present at high frequency in HTNV-immune individuals many years after acute infection. The identified epitopes may be useful for the design of new HTNV vaccine and the understanding of CTL response to HTNV during acute infection. This work was supported by grants from the National Natural Science Foundation of China (No.30471625).
Protective and Pathologic roles of the Immune Response to Mouse Hepatitis Virus-1 (MHV-1): Implications for Severe Acute Respiratory Syndrome (128.8)
Authors: Khanolkar Aaruni , Hartwig Stacey M , Pewe Lecia L , Varga Steven M , Harty John T
Published: January 2023

Intranasal MHV-1 infection of mice induces lung pathology similar to that observed in SARS patients. However the severity of MHV-1-induced pulmonary disease varies among mouse strains and it has been suggested that differences in the host immune response might account for this variation. It has also been suggested that immunopathology may represent an important clinical feature of SARS. Little is known about the host immune response to MHV-1 and how it might contribute to some of the pathological changes detected in infected mice. Here we show that an intact Type 1 IFN system, NK cells and the adaptive immune responses are important contributors to the resistance of B6 mice to MHV-1 induced disease. In A/J and C3H/HeJ mice, that are highly susceptible to MHV-1-induced disease, we demonstrate that both CD4 and CD8 T cells contribute to morbidity during primary infection, and memory responses can enhance morbidity and mortality during subsequent re-exposure to MHV-1. However, morbidity in A/J and C3H/HeJ mice can be minimized by treating them with immune serum prior to MHV-1 infection. Overall our findings highlight the role of the host immune response in contributing to the pathogenesis of coronavirus-induced respiratory disease, and have relevant implications for the design of rational vaccine strategies to combat such infections.