bluuebunny/crossref_metadata_2025 huggingface dataset:
Repression of IL-2 Promoter Activity by the Novel Basic Leucine Zipper p21SNFT Protein
Authors: Iacobelli Milena , Wachsman William , McGuire Kathleen L.
Published: April 2014

IL-2 is the major autocrine and paracrine growth factor produced by T cells upon T cell stimulation. The inducible expression of IL-2 is highly regulated by multiple transcription factors, particularly AP-1, which coordinately activate the promoter. Described here is the ability of the novel basic leucine zipper protein p21SNFT to repress AP-1 activity and IL-2 transcription. A detailed analysis of the repression by p21SNFT repression on the IL-2 promoter distal NF-AT/AP-1 site demonstrates that it can bind DNA with NF-AT and Jun, strongly suggesting that it represses NF-AT/AP-1 activity by competing with Fos proteins for Jun dimerization. The importance of this repression is that p21SNFT inhibits the trans-activation potential of protein complexes that contain Jun, thereby demonstrating an additional level of control for the highly regulated, ubiquitous AP-1 transcription factor and the IL-2 gene.
Identification of a Novel Tumor-Specific CTL Epitope Presented by RMA, EL-4, and MBL-2 Lymphomas Reveals Their Common Origin
Authors: van Hall Thorbald , van Bergen Jeroen , van Veelen Peter A. , Kraakman Margriet , Heukamp Lukas C. , Koning Frits , Melief Cornelis J. M. , Ossendorp Ferry , Offringa Rienk
Published: April 2014

C57BL/6 mice generate a vigorous H-2Db-restricted CTL response against murine leukemia virus (MuLV)-induced tumors. For many years it has been suggested that this response is directed to an MuLV-encoded peptide as well as to a nonviral tumor-associated peptide. Recently, a peptide from the leader sequence of gag was demonstrated to be the MuLV-derived epitope. Here we describe the molecular identification of the tumor-associated epitope. Furthermore, we show that the CTL response against this epitope can restrict the outgrowth of MuLV-induced tumors in vivo. The epitope is selectively presented by the MuLV-induced T cell tumors RBL-5, RMA, and MBL-2 as well as by the chemically induced T cell lymphoma EL-4. Intriguingly, these tumors share expression of the newly identified epitope because they represent variants of the same clonal tumor cell line, as evident from sequencing of the TCR α- and β-chains, which proved to be identical. Our research shows that all sources of RBL-5, RMA, RMA-S, MBL-2, and EL-4 tumors are derived from a single tumor line, most likely EL-4.
Interaction of C1q and Mannan-Binding Lectin (MBL) with C1r, C1s, MBL-Associated Serine Proteases 1 and 2, and the MBL-Associated Protein MAp19
Authors: Thiel Steffen , Petersen Steen V. , Vorup-Jensen Thomas , Matsushita Misao , Fujita Teizo , Stover Cordula M. , Schwaeble Wilhelm J. , Jensenius Jens C.
Published: April 2014

Mannan-binding lectin (MBL) and C1q activate the complement cascade via attached serine proteases. The proteases C1r and C1s were initially discovered in a complex with C1q, whereas the MBL-associated serine proteases 1 and 2 (MASP-1 and -2) were discovered in a complex with MBL. There is controversy as to whether MBL can utilize C1r and C1s or, inversely, whether C1q can utilize MASP-1 and 2. Serum deficient in C1r produced no complement activation in IgG-coated microwells, whereas activation was seen in mannan-coated microwells. In serum, C1r and C1s were found to be associated only with C1q, whereas MASP-1, MASP-2, and a third protein, MAp19 (19-kDa MBL-associated protein), were found to be associated only with MBL. The bulk of MASP-1 and MAp19 was found in association with each other and was not bound to MBL or MASP-2. The interactions of MASP-1, MASP-2, and MAp19 with MBL differ from those of C1r and C1s with C1q in that both high salt concentrations and calcium chelation (EDTA) are required to fully dissociate the MASPs or MAp19 from MBL. In the presence of calcium, most of the MASP-1, MASP-2, and MAp19 emerged on gel-permeation chromatography as large complexes that were not associated with MBL, whereas in the presence of EDTA most of these components formed smaller complexes. Over 95% of the total MASPs and MAp19 found in serum are not complexed with MBL.
Treatment of Human B Cell Lymphoma Xenografts with a CD3 × CD19 Diabody and T Cells
Authors: Cochlovius Björn , Kipriyanov Sergey M. , Stassar Marike J. J. G. , Christ Oliver , Schuhmacher Jochen , Strauß Gudrun , Moldenhauer Gerhard , Little Melvyn
Published: April 2014

The use of anti-CD3 × antitumor bispecific Abs is an attractive and highly specific approach in cancer therapy. Recombinant Ab technology now provides powerful tools to enhance the potency of such immunotherapeutic constructs. We designed a heterodimeric diabody specific for human CD19 on B cells and CD3ε chain of the TCR complex. After production in Escherichia coli and purification, we analyzed its affinity, stability, and pharmacokinetics, and tested its capacity to stimulate T cell proliferation and mediate in vitro lysis of CD19+ tumor cells. The effect of the diabody on tumor growth was investigated in an in vivo model using immunodeficient mice bearing a human B cell lymphoma. The CD3 × CD19 diabody specifically interacted with both CD3- and CD19-positive cells, was able to stimulate T cell proliferation in the presence of tumor cells, and induced the lysis of CD19+ cells in the presence of activated human PBL. The lytic potential of the diabody was enhanced in the presence of an anti-CD28 mAb. In vivo experiments indicated a higher stability and longer blood retention of diabodies compared with single chain Fv fragments. Treatment of immunodeficient mice bearing B lymphoma xenografts with the diabody and preactivated human PBL efficiently inhibited tumor growth. The survival time was further prolonged by including the anti-CD28 mAb. The CD3 × CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.
IL-12 Is Required to Maintain a Th1 Response DuringLeishmania majorInfection
Authors: Park Audrey Y. , Hondowicz Brian D. , Scott Phillip
Published: April 2014

IL-12 initiates Th1 cell development and cell-mediated immunity, but whether IL-12 contributes to the maintenance of a Th1 response is unclear. To address this question, we infected IL-12 p40−/− C57BL/6 mice with Leishmania major, an intracellular protozoan parasite controlled by a cell-mediated immune response, and simultaneously administered IL-12. Whereas untreated p40−/− mice developed an uncontrolled infection, p40−/− mice treated with IL-12 for the first 2 or 4 wk of infection developed a Th1 response and resolved their lesions. However, the induction of this protective Th1 cell response by IL-12 treatment was not associated with long term immunity. We observed that on rechallenge in the absence of IL-12, the mice exhibited a susceptible phenotype. In addition, without rechallenge, lesions in the IL-12-treated p40−/− mice developed several weeks after cessation of IL-12 treatment. In both cases, disease was associated with the loss of a Th1 response and the development of a Th2 response. Our observations are not limited to the C57BL/6 strain, because IL-12 treatment was also unable to provide lasting protection to p40−/− BALB/c mice. Finally, we found that although Th1 cells from healed wild-type C57BL/6 mice adoptively transferred protection to L. major-infected RAG−/− mice, they were unable to protect p40−/− mice. In conclusion, these studies provide the first demonstration that IL-12 is required not only to initiate Th1 cell development but also throughout infection to maintain a Th1 cell response and resistance to L. major.
InvasiveShigella flexneriActivates NF-κB Through a Lipopolysaccharide-Dependent Innate Intracellular Response and Leads to IL-8 Expression in Epithelial Cells
Authors: Philpott Dana J. , Yamaoka Shoji , Israël Alain , Sansonetti Philippe J.
Published: April 2014

The pathogenesis of Shigella flexneri infection centers on the ability of this organism to invade epithelial cells and initiate an intense inflammatory reaction. Because NF-κB is an important transcriptional regulator of genes involved in inflammation, we investigated the role of this transcription factor during S. flexneri infection of epithelial cells. Infection of HeLa cells with invasive S. flexneri induced NF-κB DNA-binding activity; noninvasive S. flexneri strains did not lead to this activation. The pathway leading to NF-κB activation by invasive S. flexneri involved the kinases, NF-κB-inducing kinase, IκB kinase-1, and IκB kinase-2. NF-κB activation was linked to inflammation, because invasive S. flexneri activated an IL-8 promoter-driven reporter gene, and the κB site within this promoter was indispensable for its induction. Microinjection of bacterial culture supernatants into HeLa cells suggested that LPS is responsible for NF-κB activation by S. flexneri infection. In conclusion, the eukaryotic transcription factor NF-κB was activated during S. flexneri infection of epithelial cells, which suggests a role for this transcriptional regulator in modulating the immune response during infection in vivo.
Mycobacterium tuberculosis-Reactive CD8+ T Lymphocytes: The Relative Contribution of Classical Versus Nonclassical HLA Restriction
Authors: Lewinsohn David M. , Briden Andria L. , Reed Steven G. , Grabstein Kenneth H. , Alderson Mark R.
Published: April 2014

Previous studies in mice and humans models have suggested an important role for CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). In humans, CD8+ Mtb-reactive T cells have been described that are HLA-A2-, B52-, as well as CD1-restricted. Recently, we have described Mtb-specific CD8+ T cells that are neither HLA-A-, B-, or C- nor group 1 CD1-restricted. At present, little is known about the relative contribution of each of these restriction specificities to the overall CD8+ response to Mtb. An IFN-γ enzyme-linked immunospot assay was used to determine the frequency of Mtb-reactive CD8+ T cells directly from PBMC. The effector cell frequency among five healthy purified protein derivative-positive subjects was 1/7,600 ± 4,300 compared with 1/16,000 ± 7,000 in six purified protein derivative-negative controls. To determine the frequencies of classically, CD1-, and nonclassically restricted cells, a limiting dilution analysis was performed. In one purified protein derivative-positive subject, 192 clones were generated using Mtb-infected dendritic cells (DC). Clones were assessed for reactivity against control autologous DC, Mtb-infected autologous DC, and HLA-mismatched CD1+ targets (DC), as well as HLA-mismatched CD1− targets (macrophages). Of the 96 Mtb-reactive CD8+ T cell clones, four (4%) were classically restricted and 92 (96%) were nonclassically restricted. CD1-restricted cells were not detected. Of the classically restricted cells, two were HLA-B44 restricted and one was HLA-B14 restricted. These results suggest that while classically restricted CD8+ lymphocytes can be detected, they comprise a relatively small component of the overall CD8+ T cell response to Mtb. Further definition of the nonclassical response may aid development of an effective vaccine against tuberculosis.
Identification of NY-ESO-1 Peptide Analogues Capable of Improved Stimulation of Tumor-Reactive CTL
Authors: Chen Ji-Li , Dunbar P. Rod , Gileadi Uzi , Jäger Elke , Gnjatic Sacha , Nagata Yasuhiro , Stockert Elisabeth , Panicali Dennis L. , Chen Yao-Tseng , Knuth Alexander , Old Lloyd J. , Cerundolo Vincenzo
Published: April 2014

Expression of NY-ESO-1 in a high proportion of different human tumors makes this protein a very attractive vaccine target. NY-ESO-1 peptides, recognized by HLA-A2-restricted CTL, have recently been described. However, it remains unclear how efficiently tumors generate these epitopes, and whether peptide analogues can be used for optimal expansion and activation of NY-ESO-1-specific HLA-A2-restricted CTL. By generating unique CTL clones, we demonstrate that NY-ESO-1-positive tumor cells are efficiently killed by HLA-A2-restricted CTL specific for the peptide epitope NY-ESO-1 157–165. Presentation of this epitope is not affected by the presence or absence of the proteasome subunits low molecular proteins 2 and 7 and is not blocked by proteasome inhibitors, while it is impaired in the TAP-deficient cell line LBL 721.174. NY-ESO-1 157–165 peptide analogues were compared for their antigenicity and immunogenicity using PBL from melanoma patients. Three peptides, containing the carboxyl-terminal cysteine substituted for either valine, isoleucine, or leucine, were recognized at least 100 times more efficiently than the wild-type peptide by specific CTL. Peptide analogues were capable of stimulating the expansion of NY-ESO-1-specific CTL from PBL of melanoma patients much more efficiently than wild-type peptide. These findings define the processing requirements for the generation of the NY-ESO-1 157–165 epitope. Identification of highly antigenic NY-ESO-1 peptide analogues may be important for the development of vaccines capable of expanding NY-ESO-1-specific CTL in cancer patients.
Macrophage Inflammatory Protein-1α Is a Critical Mediator of Host Defense Against Invasive Pulmonary Aspergillosis in Neutropenic Hosts
Authors: Mehrad Borna , Moore Thomas A. , Standiford Theodore J.
Published: April 2014

Invasive pulmonary aspergillosis is a devastating complication of immunosuppression that usually occurs in neutropenic patients. In this setting, augmentation of the antifungal activity of available immune cells may improve the outcome of the infection. Macrophage inflammatory protein-1α (MIP-1α) is a CC chemokine with potent chemotactic activity for various subsets of mononuclear leukocytes. We therefore tested the hypothesis that the influx of mononuclear cells into the lung in invasive pulmonary aspergillosis is in part mediated by MIP-1α, and the manipulation of this ligand alters the outcome of the infection. We found that in both immunocompetent and neutropenic mice, MIP-1α was induced in the lungs in response to intratracheal administration of Aspergillus fumigatus conidia. In neutrophil-depleted mice challenged with intratracheal conidia, there was evidence of invasive fungal pneumonia associated with a predominantly mononuclear leukocyte infiltrate. Ab-mediated depletion of MIP-1α resulted in a 6-fold increase in mortality in neutropenic mice, which was associated with a 12-fold increase in lung fungal burden. Studies of single-cell suspensions of whole lungs revealed a 36% decrease in total lung leukocyte infiltration as a result of MIP-1α neutralization. Flow cytometry on whole lung suspensions showed a 41% reduction in lung monocyte/macrophages as a result of MIP-1α neutralization, but no difference in other lung leukocyte subsets. These studies indicate that MIP-1α is a critical mediator of host defense against A. fumigatus in the setting of neutropenia and may be an important target in devising future therapeutic strategies against invasive aspergillosis.
IL-10 Converts Human Dendritic Cells into Macrophage-Like Cells with Increased Antibacterial Activity Against VirulentMycobacterium tuberculosis
Authors: Förtsch Daniela , Röllinghoff Martin , Stenger Steffen
Published: April 2014

Dendritic cells (DC) are unique in their ability to initiate a primary immune response by the presentation of soluble Ags to T cells. Recent studies have shown that DC also phagocytose particulate Ags including the intracellular pathogen Mycobacterium tuberculosis. However, it is not known whether DC contain the growth of intracellular organisms or allow unlimited replication. To address this question, we infected human DC with a virulent strain of M. tuberculosis and monitored the intracellular growth. The bacteria grew two orders of magnitude within 7 days of culture. Among cytokines known to modulate mycobacterial growth particularly in murine macrophages (TNF-α, IFN-γ, TGF-β, IL-4), only IL-10 modulated the growth in human DC. This effect was specific for immature dendritic cells, as IL-10 did not induce growth inhibition in human macrophages. In searching for the mechanism of growth inhibition, we found that IL-10 induces the down-regulation of the DC marker CD1, while the macrophage marker CD14 was up-regulated. Functionally, IL-10-treated cells had a reduced capacity to induce an alloresponse, but phagocytic uptake of M. tuberculosis was more efficient. We also show that DC are inferior to macrophages in containing mycobacterial growth. These findings show that IL-10 converts DC into macrophage-like cells, thereby inducing the growth inhibition of an intracellular pathogen. At the site of a local immune response, such as a tuberculous granuloma, IL-10 might therefore participate in the composition of the cellular microenvironment by affecting the maturity and function of DC.
Nitric Oxide Induces Thymocyte Apoptosis Via a Caspase-1-Dependent Mechanism
Authors: Zhou Xin , Gordon Sherilyn A. , Kim Young-Myeong , Hoffman Rosemary A. , Chen Yue , Zhang Xiao-Ru , Simmons Richard L. , Ford Henri R.
Published: April 2014

We previously showed that NO induces apoptosis in thymocytes via a p53-dependent pathway. In the present study, we investigated the role of caspases in this process. The pan-caspase inhibitor, ZVAD-fmk, and the caspase-1 inhibitor, Ac-YVAD-cho, both inhibited NO-induced thymocyte apoptosis in a dose-dependent manner, whereas the caspase-3 inhibitor, Ac-DEVD-cho, had little effect even at concentrations up to 500 μM. ZVAD-fmk and Ac-YVAD-cho were able to inhibit apoptosis when added up to 12 h, but not 16 h, after treatment with the NO donor S-nitroso-N-acetyl penicillamine (SNAP). Caspase-1 activity was up-regulated at 4 h and 8 h and returned to baseline by 24 h; caspase-3 activity was not detected. Cytosolic fractions from SNAP-treated thymocytes cleaved the inhibitor of caspase-activated deoxyribonuclease. Such cleavage was completely blocked by Ac-YVAD-cho, but not by Ac-DEVD-cho or DEVD-fmk. Poly(ADP-ribose) polymerase (PARP) was also cleaved in thymocytes 8 h and 12 h after SNAP treatment; addition of Ac-YVAD-cho to the cultures blocked PARP cleavage. Furthermore, SNAP induced apoptosis in 44% of thymocytes from wild-type mice; thymocytes from caspase-1 knockout mice were more resistant to NO-induced apoptosis. These data suggest that NO induces apoptosis in thymocytes via a caspase-1-dependent but not caspase-3-dependent pathway. Caspase-1 alone can cleave inhibitor of caspase-activated deoxyribonuclease and lead to DNA fragmentation, thus providing a novel pathway for NO-induced thymocyte apoptosis.
Glycosyl-Phosphatidylinositol Reanchoring Unmasks Distinct Antigen-Presenting Pathways for CD1b and CD1c
Authors: Geho David H. , Fayen John D. , Jackman Robin M. , Moody D. Branch , Porcelli Steven A. , Tykocinski Mark L.
Published: April 2014

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b · DAF and CD1c · DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c · DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b · DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c · DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b · DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.
The Alloreactive and Self-Restricted CD4+ T Cell Response Directed Against a Single MHC Class II/Peptide Combination
Authors: Kovalik Jean-Paul , Singh Nagendra , Mendiratta Sanjeev K. , Martin W. David , Ignatowicz Leszek , Van Kaer Luc
Published: April 2014

The cellular basis for allograft rejection derives from the strong T cell response to cells bearing foreign MHC. While it was originally assumed that alloreactive T cells focus their recognition on the polymorphic residues that differ between syngeneic and allogeneic MHC molecules, studies with MHC class I-restricted CTL have shown that MHC-bound peptides play a critical role in allorecognition. It has been suggested that alloreactive T cells depend more strongly on interactions with the MHC molecule than with the associated peptide, but there is little evidence to support this idea. Here we have studied the alloreactive and self-restricted response directed against the class II H2-Ab molecule bound with a single peptide, Ep, derived from the H2-Eα chain. This MHC class II-peptide combination was a poor target and stimulator of alloreactive CD4+ T cell responses, indicating that MHC-bound peptides are as important for alloreactive CD4+ T cells as they are for alloreactive CTL. We also generated alloreactive T cells with exquisite specificity for the Ab/Ep complex, and compared their reactivity with self-restricted T cells specific for the same Ab/Ep complex. Our results showed that peptide-specific alloreactive T cells, as compared with self-restricted T cells, were more sensitive to peptide stimulation, but equally sensitive to amino acid substitutions in the peptide. These findings indicate that alloreactive and self-restricted T cells interact similarly with their MHC/peptide ligand.
Src Homology Region 2 (SH2) Domain-Containing Phosphatase-1 Dephosphorylates B Cell Linker Protein/SH2 Domain Leukocyte Protein of 65 kDa and Selectively Regulates c-Jun NH2-Terminal Kinase Activation in B Cells
Authors: Mizuno Kazuya , Tagawa Yuko , Mitomo Katsuyuki , Arimura Yutaka , Hatano Norikazu , Katagiri Tatsuo , Ogimoto Mami , Yakura Hidetaka
Published: April 2014

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.
Functional Involvement of E-Cadherin in TGF-β1-Induced Cell Cluster Formation of In Vitro Developing Human Langerhans-Type Dendritic Cells
Authors: Riedl Elisabeth , Stöckl Johannes , Majdic Otto , Scheinecker Clemens , Rappersberger Klemens , Knapp Walter , Strobl Herbert
Published: April 2014

Epithelial Langerhans cells (LC) represent immature dendritic cells that require TGF-β1 stimulation for their development. Little is known about the mechanisms regulating LC generation from their precursor cells. We demonstrate here that LC development from human CD34+ hemopoietic progenitor cells in response to TGF-β1 costimulation (basic cytokine combination GM-CSF plus TNF-α, stem cell factor, and Flt3 ligand) is associated with pronounced cell cluster formation of developing LC precursor cells. This cell-clustering phenomenon requires hemopoietic progenitor cell differentiation, since it is first seen on day 4 after culture initiation of CD34+ cells. Cell cluster formation morphologically indicates progenitor cell development along the LC pathway, because parallel cultures set up in the absence of exogenous TGF-β1 fail to form cell clusters and predominantly give rise to monocyte, but not LC, development (CD1a−, lysozyme+, CD14+). TGF-β1 costimulation of CD34+ cells induces neoexpression of the homophilic adhesion molecule E-cadherin in the absence of the E-cadherin heteroligand CD103. Addition of anti-E-cadherin mAb or mAbs to any of the constitutively expressed adhesion molecule (CD99, CD31, LFA-1, or CD18) to TGF-β1-supplemented progenitor cell cultures inhibits LC precursor cell cluster formation, and this effect is, with the exception of anti-E-cadherin mAb, associated with inhibition of LC generation. Addition of anti-E-cadherin mAb to the culture allows cell cluster-independent generation of LC from CD34+ cells. Thus, functional E-cadherin expression and homotypic cell cluster formation represent a regular response of LC precursor cells to TGF-β1 stimulation, and cytoadhesive interactions may modulate LC differentiation from hemopoietic progenitor cells.
Bone-Marrow Chimeras Reveal Hemopoietic and Nonhemopoietic Control of Resistance to Experimental Lyme Arthritis
Authors: Brown Charles R. , Reiner Steven L.
Published: April 2014

Both genetic resistance and susceptibility to development of experimental Lyme arthritis are mediated by the innate immune response. To determine whether this process is mainly controlled by hemopoietic or nonhemopoietic cells, we created bone marrow (BM) chimeric mice between arthritis-resistant DBA/2J (DBA) and arthritis-susceptible C3H/HeJ (C3H) mice and infected them with Borrelia burgdorferi. Both sets of BM chimeric mice, C3H donors into DBA recipients (C→D) and DBA donors into C3H recipients (D→C), as well as DBA sham chimeric mice (D→D) were resistant to the development of experimental Lyme arthritis as measured by ankle swelling and arthritis severity scores. Only the C3H sham chimeric mice (C→C) developed severe arthritis. These results indicate that independent and nonoverlapping mechanisms exist in hemopoietic and nonhemopoietic cellular compartments that can provide protection against arthritic pathology.
Pulmonary Mononuclear Cell Responses to Antigens ofMycobacterium tuberculosisin Healthy Household Contacts of Patients with Active Tuberculosis and Healthy Controls from the Community
Authors: Schwander Stephan K. , Torres Martha , Carranza C Claudia , Escobedo Dante , Tary-Lehmann Magdalena , Anderson Peter , Toossi Zahra , Ellner Jerrold J. , Rich Elizabeth A. , Sada Eduardo
Published: April 2014

Protective immunity against Mycobacterium tuberculosis requires CD4+ lymphocyte-mediated immune responses and IFN-γ activity. As the primary portal of entry of M. tuberculosis is the lung, pulmonary immune responses against multiple M. tuberculosis Ags were compared between both M. tuberculosis-exposed tuberculin skin test-positive healthy household contacts (HHC) of patients with active sputum smear and culture-positive tuberculosis and tuberculin skin test-positive healthy control individuals from the community (CC). Frequencies of M. tuberculosis Ag-specific IFN-γ-producing cells, IFN-γ concentrations in culture supernatants, and DNA synthesis in bronchoalveolar cells (BAC) and PBMC were studied in HHC (n = 10) and CC (n = 15). Using enzyme-linked immunospot assay we found higher frequencies of IFN-γ-producing cells with specificity to M. tuberculosis-secreted Ag 85 (Ag 85) in BAC from HHC than in BAC from CC (p < 0.022) and relative to autologous PBMC, indicating compartmentalization of Ag 85-specific cells to the lungs. Further, IFN-γ-producing cells with specificity to components A and B of Ag 85 were specifically compartmentalized to the lungs in HHC (p < 0.05). IFN-γ concentrations in culture supernatants of BAC and Ag-specific DNA synthesis were low and comparable in the two subject groups. Increased immune responses to Ag 85 at the site of repeated exposure to M. tuberculosis (the lung) may represent an important component of protective immunity against M. tuberculosis. Correlates of protective immunity against M. tuberculosis are required for assessment of the efficiency of anti-tuberculous vaccines.
Urokinase Receptor-Deficient Mice Have Impaired Neutrophil Recruitment in Response to PulmonaryPseudomonas aeruginosaInfection
Authors: Gyetko Margaret R. , Sud Sudha , Kendall Tara , Fuller Jennifer A. , Newstead Michael W. , Standiford Theodore J.
Published: April 2014

Leukocytes express both urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). Evidence in vitro has implicated uPAR as a modulator of β2 integrin function, particularly CR3 (CD11b/CD18, Mac-1). Pseudomonas aeruginosa infection has been demonstrated to recruit neutrophils to the pulmonary parenchyma by a β2 integrin-dependent mechanism. We demonstrate that mice deficient in uPAR (uPAR−/−) have profoundly diminished neutrophil recruitment in response to P. aeruginosa pneumonia compared with wild-type (WT) mice. The requirement for uPAR in neutrophil recruitment is independent of the serine protease uPA, as neutrophil recruitment in uPA−/− mice is indistinguishable from recruitment in WT mice. uPAR−/− mice have impaired clearance of P. aeruginosa compared with WT mice, as demonstrated by CFU and comparative histology. WT mice have diminished neutrophil recruitment to the lung when an anti-CD11b mAb is given before inoculation with the pathogen, while recruitment of uPAR−/− neutrophils is unaffected. We conclude that uPAR is required for the recruitment of neutrophils to the lung in response to P. aeruginosa pneumonia and that this requirement is independent of uPA. Further, we show that uPAR and CR3 act by a common mechanism during neutrophil recruitment to the lung in response to P. aeruginosa. This is the first report of a requirement for uPAR during cellular recruitment in vivo against a clinically relevant pathogen.
Chloroquine Interferes with Lipopolysaccharide-Induced TNF-α Gene Expression by a Nonlysosomotropic Mechanism
Authors: Weber Stephen M. , Levitz Stuart M.
Published: April 2014

Chloroquine (CQ) is a lysosomotropic weak base with over 60 years of clinical use for the treatment of malaria and rheumatologic disorders. Consistent with its anti-inflammatory properties, CQ has been shown to interfere with TNF-α release from mononuclear phagocytes. Because it is unclear how CQ mediates these immunomodulatory effects, we set out to elucidate its mechanism of action. CQ exhibited dose-dependent inhibition of LPS-induced TNF-α release from human PBMC at therapeutically attainable concentrations. Additional studies to determine the specificity of this effect showed that although CQ reduced IL-1β and IL-6 release, secretion of RANTES was unaffected. CQ acted by reducing TNF-α mRNA accumulation without destabilizing its mRNA or interfering with NF-κB nuclear translocation or p50/p65 isoform composition of DNA-binding complexes. Intracellular cytokine staining indicated that CQ reduced TNF-α production pretranslationally without interfering with TNF-α processing or release. We utilized bafilomycin A1 pretreatment to block the pH-dependent trapping of CQ in endosomes and lysosomes. Although bafilomycin A1 alone did not interfere with TNF-α expression, preincubation augmented the ability of CQ to reduce TNF-α mRNA levels, suggesting that CQ did not act by a lysosomotropic mechanism. Using confocal microscopy, we showed that bafilomycin A1 pretreatment resulted in a dramatic redistribution of quinacrine, a fluorescent congener of CQ, from cytoplasmic vacuoles to the nucleus. These data indicate that CQ inhibits TNF-α gene expression without altering translocation of NF-κB p50/p65 heterodimers. This dose-dependent effect occurs over a pharmacologically relevant concentration range and does not require pH-dependent lysosomotropic accumulation of CQ.
CXCR3 Expression and Activation of Eosinophils: Role of IFN-γ-Inducible Protein-10 and Monokine Induced by IFN-γ
Authors: Jinquan Tan , Jing Chen , Jacobi Henrik H. , Reimert Claus M. , Millner Anders , Quan Sha , Hansen Jens B. , Dissing Steen , Malling Hans-Jørgen , Skov Per S. , Poulsen Lars K.
Published: April 2014

CXC chemokine receptor 3 (CXCR3), predominately expressed on memory/activated T lymphocytes, is a receptor for both IFN-γ-inducible protein-10 (γ IP-10) and monokine induced by IFN-γ (Mig). We report a novel finding that CXCR3 is also expressed on eosinophils. γ IP-10 and Mig induce eosinophil chemotaxis via CXCR3, as documented by the fact that anti-CXCR3 mAb blocks γ IP-10- and Mig-induced eosinophil chemotaxis. γ IP-10- and Mig-induced eosinophil chemotaxis are up- and down-regulated by IL-2 and IL-10, respectively. Correspondingly, CXCR3 protein and mRNA expressions in eosinophils are up- and down-regulated by IL-2 and IL-10, respectively, as detected using flow cytometry, immunocytochemical assay, and a real-time quantitative RT-PCR technique. γ IP-10 and Mig act eosinophils to induce chemotaxis via the cAMP-dependent protein kinase A signaling pathways. The fact that γ IP-10 and Mig induce an increase in intracellular calcium in eosinophils confirms that CXCR3 exists on eosinophils. Besides induction to chemotaxis, γ IP-10 and Mig also activate eosinophils to eosinophil cationic protein release. These results indicate that CXCR3-γ IP-10 and -Mig receptor-ligand pairs as well as the effects of IL-2 and IL-10 on them may be especially important in the cytokine/chemokine environment for the pathophysiologic events of allergic inflammation, including initiation, progression, and termination in the processes.
Heterogeneous Effects of IL-2 on Collagen-Induced Arthritis
Authors: Thornton Sherry , Boivin Gregory P. , Kim Kwang N. , Finkelman Fred D. , Hirsch Raphael
Published: April 2014

IL-2 is generally considered a pro-inflammatory cytokine that exacerbates Th1-mediated disease states, such as autoimmune arthritis. Consistent with this role for IL-2, recent studies from our laboratory demonstrate that IL-2 mRNA is markedly increased during the acute stage of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. To further define the role of IL-2 in CIA, the levels of IL-2 protein and its receptor and the effects of IL-2 administration were analyzed during CIA. IL-2 protein and IL-2R were preferentially expressed at disease onset, compared with later stages of disease. Administration of recombinant human IL-2 (rhIL-2) at, or just before, disease onset exacerbated disease; surprisingly, rhIL-2 given before disease onset inhibited CIA, associated with reduced cellular and humoral responses to type II collagen. Determination of in vivo serum levels of Th1 and Th2 cytokines in response to rhIL-2 treatment demonstrated that IFN-γ, but not IL-4, was markedly up-regulated in response to IL-2. In mice treated with anti-IFN-γ Ab, both early and late IL-2 administration exacerbated CIA. Thus, IL-2 can have two opposite effects on autoimmune arthritis, a direct stimulatory effect and an indirect suppressive effect that is mediated by IFN-γ.
Airway Remodeling Is Absent in CCR1−/− Mice During Chronic Fungal Allergic Airway Disease
Authors: Blease Kate , Mehrad Borna , Standiford Theodore J. , Lukacs Nicholas W. , Kunkel Steven L. , Chensue Stephen W. , Lu Bao , Gerard Craig J. , Hogaboam Cory M.
Published: April 2014

Asthmatic-like reactions characterized by elevated IgE, Th2 cytokines, C-C chemokines, eosinophilic inflammation, and persistent airway hyperresponsiveness follow pulmonary exposure to the spores or conidia from Aspergillus fumigatus fungus in sensitized individuals. In addition to these features, subepithelial fibrosis and goblet cell hyperplasia characterizes fungal-induced allergic airway disease in mice. Because lung concentrations of macrophage inflammatory protein-1α and RANTES were significantly elevated after A. fumigatus-sensitized mice received an intrapulmonary challenge with A. fumigatus spores or conidia, the present study addressed the role of their receptor, C-C chemokine receptor 1 (CCR1), in this model. A. fumigatus-sensitized CCR1 wild-type (+/+) and CCR1 knockout (−/−) mice exhibited similar increases in serum IgE and polymorphonuclear leukocyte numbers in the bronchoalveolar lavage. Airway hyperresponsiveness was prominent in both groups of mice at 30 days after an intrapulmonary challenge with A. fumigatus spores or conidia. However, whole lung levels of IFN-γ were significantly higher whereas IL-4, IL-13, and Th2-inducible chemokines such as C10, eotaxin, and macrophage-derived chemokine were significantly lower in whole lung samples from CCR1−/− mice compared with CCR1+/+ mice at 30 days after the conidia challenge. Likewise, significantly fewer goblet cells and less subepithelial fibrosis were observed around large airways in CCR1−/− mice at the same time after the conidia challenge. Thus, these findings demonstrate that CCR1 is a major contributor to the airway remodeling responses that arise from A. fumigatus-induced allergic airway disease.
Early Local Generation of C5a Initiates the Elicitation of Contact Sensitivity by Leading to Early T Cell Recruitment
Authors: Tsuji Ryohei F. , Kawikova Ivana , Ramabhadran Rajani , Akahira-Azuma Moe , Taub Dennis , Hugli Tony E. , Gerard Craig , Askenase Philip W.
Published: April 2014

We have shown previously that an early complement C5-dependent cascade is required to recruit T cells to elicit 24-h contact sensitivity (CS) responses. In this paper, we have characterized molecular events of this early required cascade by biochemically analyzing extracts of mouse ears undergoing elicitation of CS. Chemotactic activity was found after local Ag challenge, in CS ear extracts early (by 1 h), in CS ear extracts late (through 24 h), in previously immunized mice, but not in ears of vehicle-immunized or non-immune-challenged mice. The early chemotactic activity at 2 h was likely caused by C5a, because it was neutralized in vitro by anti-C5a Ab, was inactive on C5aR-deficient (C5aR−/−) macrophages, and was absent in C5-deficient mice. The activity was present in T cell-deficient mice, but elaboration was Ag-specific. This T cell-independent, Ag-specific elaboration of C5a early in CS ear responses likely led to T cell recruitment, because subsequent local IFN-γ mRNA and protein expression, as markers of T cell arrival and activation, began by 4 h after Ag challenge. In contrast to early C5a chemotactic activity, late chemotactic activity 24 h after Ag challenge was unaffected by anti-C5, was active on C5aR−/− macrophages, was T cell-dependent, and by ELISA appeared largely due to chemokines (macrophage-inflammatory protein-1α and -1β, IFN-γ-inducible protein-10, and monocyte chemoattractant protein-1). Importantly, early generation of C5a was required for T cell recruitment because C5aR−/− mice had absent 24-h CS. Taken together, these findings indicate an important linkage of C5a as a component of early activated innate immunity that is required for later elicitation of acquired T cell immunity, probably by facilitating the initial recruitment of T cells into the Ag-challenged local site in CS responses.
Phenotypic and Functional Maturation of Dendritic Cells Mediated by Heparan Sulfate
Authors: Kodaira Yuzo , Nair Smita K. , Wrenshall Lucile E. , Gilboa Eli , Platt Jeffrey L.
Published: April 2014

Primary immune responses are thought to be induced by dendritic cells. To promote such responses, dendritic cells must be activated by exogenous agonists, such as LPS, or by products of activated leukocytes, such as TNF-α and IL-1. How dendritic cells might be activated in the absence of exogenous stimuli, or without the immediate presence of activated leukocytes, as might occur in immunity to tumor cells or transplants, is unknown. We postulated that heparan sulfate, an acidic, biologically active polysaccharide associated with cell membranes and extracellular matrices, which is rapidly released under conditions of inflammation and tissue damage, might provide such a stimulus. Incubation of immature murine dendritic cells with heparan sulfate induced phenotypic maturation evidenced by up-regulation of I-A, CD40, CD54 (ICAM-1), CD80 (B7-1), and CD86 (B7-2). Dendritic cells exposed to heparan sulfate exhibited a markedly lowered rate of Ag uptake and increased allostimulatory capacity. Stimulation of dendritic cells with heparan sulfate induced release of TNF-α, IL-1β, and IL-6, although the maturation of dendritic cells was independent of these cytokines. These results suggest that soluble heparan sulfate chains, as products of the degradation of heparan sulfate proteoglycan, might induce maturation of dendritic cells without exogenous stimuli, thus contributing to the generation and maintenance of primary immune responses.
Regulation of Expression of Granulocyte-Macrophage Colony-Stimulating Factor in Human Bronchial Epithelial Cells: Roles of Protein Kinase C and Mitogen-Activated Protein Kinases
Authors: Reibman Joan , Talbot Anita T. , Hsu Yanshen , Ou Guoming , Jover Javier , Nilsen Diana , Pillinger Michael H.
Published: April 2014

GM-CSF has a major role in the immune and inflammatory milieu of the airway. Airway epithelial cells (AEC) are among the first targets of environmental stimuli and local cytokines, in response to which they can produce GM-CSF. The regulation of GM-CSF is only minimally understood in AEC. We hypothesized that GM-CSF expression in AEC would result from activation of protein kinase C (PKC) and subsequent activation of the extracellular signal-regulated kinase (MAPKerk1/2) pathway, so we investigated signal transduction pathways in human primary culture bronchial epithelial cells (HBECs). TNF-α, IL-1β, and PMA induced the release of GM-CSF in HBECs. The robust response to PMA was not detected in SV40 adenovirus-transformed normal human bronchial epithelial cells (BEAS-2B). PMA and TNF-α stimulation of GM-CSF required activation of PKC (inhibition by staurosporine and bisindolylmaleimide I). GM-CSF expression was up-regulated by a nonphorbol PKC activator, but not by an inactive PMA analogue. PMA-induced GM-CSF production in HBECs did not require a Ca2+ ionophore and was not inhibited by cyclosporin A. Activation of MAPKerk1/2 via PKC was associated with and was required for GM-CSF production induced by PMA and TNF-α. The data demonstrate regulation of GM-CSF in HBECs by PKC pathways converging on the MAPKerk1/2 pathway and further define cell-specific regulation critical for local airway responses.
Immune Complexes Present in the Sera of Autoimmune Mice Activate Rheumatoid Factor B Cells
Authors: Rifkin Ian R. , Leadbetter Elizabeth A. , Beaudette Britte C. , Kiani Cornelia , Monestier Marc , Shlomchik Mark J. , Marshak-Rothstein Ann
Published: April 2014

The fate of an autoreactive B cell is determined in part by the nature of the interaction of the B cell receptor with its autoantigen. In the lpr model of systemic autoimmunity, as well as in certain human diseases, autoreactive B cells expressing rheumatoid factor (RF) binding activity are prominent. A murine B cell transgenic model in which the B cell receptor is a RF that recognizes IgG2a of the j allotype (IgG2aj), but not the b allotype, was used in this study to investigate how the form of the autoantigen influences its ability to activate B cells. We found that sera from autoimmune mice, but not from nonautoimmune mice, were able to induce the proliferation of these RF+ B cells but did not stimulate B cells from RF− littermate controls. The stimulatory factor in serum was found to be IgG2aj, but the IgG2aj was stimulatory only when in the form of immune complexes. Monomeric IgG2aj failed to stimulate. Immune complexes containing lupus-associated nuclear and cytoplasmic autoantigens were particularly potent B cell activators in this system. Appropriate manipulation of such autoantibody/autoantigen complexes may eventually provide a means for therapeutic intervention in patients with certain systemic autoimmune disorders.
Expression of Human Complement Receptor 2 (CR2, CD21) inCr2−/− Mice Restores Humoral Immune Function
Authors: Marchbank Kevin J. , Watson Clay C. , Ritsema David F. , Holers V. Michael
Published: April 2014

Complement receptor type 2 (CR2, CD21) is expressed by both human and murine B cells and has been demonstrated to play a pivotal role in the humoral immune response. We have reconstituted Cr2−/− mice with an 80-kb human genomic fragment (designated P1-5) containing the full-length human CR2 (hCR2) gene. Transfection of P1-5 into the mouse A20 B cell line confirmed that it would direct expression of the hCR2 protein in mouse B cells. Immunoprecipitation analysis in these cells revealed that hCR2 coassociates with mouse CD19. After creation of transgenic mice using P1-5, we found significant expression of hCR2 on peripheral blood and splenic B cells by flow cytometric analysis. RT-PCR analysis of tissues and purified cell populations from transgene-positive mice revealed that hCR2 expression was restricted to B cells and the spleen in a pattern that matches mouse CR2. To rigorously assess the functional capabilities of hCR2, the transgene was bred onto Cr2−/− mice, which have a notable defect in response to SRBC Ag. We found that Cr2−/− mice expressing hCR2 had a substantial restoration of the humoral immune response to SRBC as compared with nontransgenic Cr2−/− littermate controls. Overall, this study suggests that hCR2 is able to substitute for mouse CR2 in the murine immune system. Therefore, hCR2-transgenic mice offer a valuable model system to further examine immunologic roles as well as structure-function relationships important for hCR2 function in primary cells in vivo.
Stat-1 Is Not Essential for Inhibition of B Lymphopoiesis by Type I IFNs
Authors: Gongora Rafael , Stephan Robert P. , Schreiber Robert D. , Cooper Max D.
Published: April 2014

Type I IFNs, IFN-α, -β, and -ω, are cytokine family members with multiple immune response roles, including the promotion of cell growth and differentiation. Conversely, the type I IFNs are potent inhibitors of IL-7-dependent growth of early B lineage progenitors, effectively aborting further B lineage differentiation at the pro-B cell stage. Type I IFNs α and β function via receptor-mediated activation of a Jak/Stat signaling pathway in which Stat-1 is functionally important, because many IFN-induced responses are abrogated in Stat-1-deficient mice. To the contrary, we show here that the inhibition of IL-7-dependent B lymphopoiesis by IFN-αβ is unaffected in Stat-1-deficient mice. The present data indicate that the type I IFNs can activate an alternative signaling pathway in which neither Stat-1 nor phosphatidylinositol 3′-kinase are essential components.