bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Regulation of Eosinophil Chemokine Signaling Via CCR3 and Non-CCR3 Pathways
Authors: Sabroe Ian , Hartnell Adele , Jopling Louise A. , Bel Sandra , Ponath Paul D. , Pease James E. , Collins Paul D. , Williams Timothy J.
Published: December 2022

To investigate eosinophil stimulation by chemokines we developed a sensitive assay of leukocyte shape change, the gated autofluorescence/forward scatter assay. Leukocyte shape change responses are mediated through rearrangements of the cellular cytoskeleton in a dynamic process typically resulting in a polarized cell and are essential to the processes of leukocyte migration from the microcirculation into sites of inflammation. We examined the actions of the chemokines eotaxin, eotaxin-2, monocyte chemoattractant protein-1 (MCP-1), MCP-3, MCP-4, RANTES, macrophage inflammatory protein-1α (MIP-1α), and IL-8 on leukocytes in mixed cell suspensions and focused on the responses of eosinophils to C-C chemokines. Those chemokines acting on CCR3 induced a rapid shape change in eosinophils from all donors; of these, eotaxin and eotaxin-2 were the most potent. Responses to MCP-4 were qualitatively different, showing marked reversal of shape change responses with agonist concentration and duration of treatment. In contrast, MIP-1α induced a potent response in eosinophils from a small and previously undescribed subgroup of donors via a non-CCR3 pathway likely to be CCR1 mediated. Incubation of leukocytes at 37°C for 90 min in the absence of extracellular calcium up-regulated responses to MCP-4 and MIP-1α in the majority of donors, and there was a small increase in responses to eotaxin. MIP-1α responsiveness in vivo may therefore be a function of both CCR1 expression levels and the regulated efficiency of coupling to intracellular signaling pathways. The observed up-regulation of MIP-1α signaling via non-CCR3 pathways may play a role in eosinophil recruitment in inflammatory states such as occurs in the asthmatic lung.
Corticotropin-Releasing Factor Receptor 1 in Mouse Spleen: Expression After Immune Stimulation and Identification of Receptor-Bearing Cells
Authors: Radulovic Marko , Dautzenberg Frank M. , Sydow Sabine , Radulovic Jelena , Spiess Joachim
Published: December 2022

A specific polyclonal Ab against the N-terminal domain of corticotropin-releasing factor (CRF) receptor, type 1 (CRF-R1), was employed to an immunohistochemical analysis of the spleen from naive mice and mice exposed to an immune challenge. Cell types stained with anti-CRF-R1 Ab were identified by their nuclear shapes and colocalization with the cell type-specific markers ER-MP58, ER-MP20, Moma-1, Moma 2, anti-CD3e mAbs, and anti-Ig Ab. Only a few clusters of CRF-R1+ cells were found in spleen sections of naive mice at sites typical for granulopoietic islands. However, a 17-fold increase in the mean number of CRF-R1+ cells was noted within hours following a challenge of acute systemic inflammation induced by i.p. administration of LPS. The majority of these cells were identified as mature neutrophils. CRF-R1 was shown to mediate suppression of the IL-1β secretion by these cells. However, at later time points a large number of granulocyte-macrophage precursors was strongly labeled with anti-CRF-R1 Ab. Western blot analysis of splenic membranes from animals treated with LPS revealed a m.w. of approximately 70,000 for CRF-R1. Subcellular staining patterns were suggestive for the predominant localization of CRF-R1 on granule membranes. CRF-R1 mRNA was detected in spleen but not in bone marrow and peripheral blood leukocytes from naive mice. Thus, it was indicated that CRF-R1 was not produced constitutively by mature or immature neutrophils. Its production was rather triggered by inflammatory stimuli.
Macrophage Inflammatory Protein-2 Is Required for Neutrophil Passage Across the Epithelial Barrier of the Infected Urinary Tract
Authors: Hang Long , Haraoka Masashi , Agace William W. , Leffler Hakon , Burdick Marie , Strieter Robert , Svanborg Catharina
Published: December 2022

IL-8 is a major human neutrophil chemoattractant at mucosal infection sites. This study examined the C-X-C chemokine response to mucosal infection, and, specifically, the role of macrophage inflammatory protein (MIP)-2, one of the mouse IL-8 equivalents, for neutrophil-epithelial interactions. Following intravesical Escherichia coli infection, several C-X-C chemokines were secreted into the urine, but only MIP-2 concentrations correlated to neutrophil numbers. Tissue quantitation demonstrated that kidney MIP-2 production was triggered by infection, and immunohistochemistry identified the kidney epithelium as a main source of MIP-2. Treatment with anti-MIP-2 Ab reduced the urine neutrophil numbers, but the mice had normal tissue neutrophil levels. By immunohistochemistry, the neutrophils were found in aggregates under the pelvic epithelium, but in control mice the neutrophils crossed the urothelium into the urine. The results demonstrate that different chemokines direct neutrophil migration from the bloodstream to the lamina propria and across the epithelium and that MIP-2 serves the latter function. These findings suggest that neutrophils cross epithelial cell barriers in a highly regulated manner in response to chemokines elaborated at this site. This is yet another mechanism that defines the mucosal compartment and differentiates the local from the systemic host response.
Induction of Autoimmunity by Multivalent Immunodominant and Subdominant T Cell Determinants of La (SS-B)
Authors: Farris A. Darise , Brown Lorena , Reynolds Pakathip , Harley John B. , James Judith A. , Scofield R. Hal , McCluskey James , Gordon Tom P.
Published: December 2022

We investigated the consequences of altering the form and valence of defined autodeterminants on the initiation and spreading of experimentally induced La/Ro autoimmunity. Anti-La and Ro (SS-A) Ab responses were monitored following immunization of healthy mice with defined immunodominant and subdominant T cell determinants of the La (SS-B) autoantigen synthesized as either monomeric or multiple antigenic (MAP) peptides. Abs to mouse La (mLa) developed faster and were of higher titer in mice immunized with the subdominant mLa25–44 MAP compared with mice immunized with the 25–44 monomer. Rapid intermolecular spreading of the autoimmune response to 60-kDa Ro was observed in AKR/J mice immunized with mLa25–44 MAP, but not in mice immunized repeatedly with monomeric peptide. A/J mice immunized and boosted with the known tolerogenic mLa287–301 determinant delivered as monomeric peptide failed to develop Abs to either intact mLa or mLa287–301 peptide. However, immunization with the multivalent mLa287–301 peptide led to the rapid production of high titer mLa autoantibodies associated with a proliferative T cell response to the mLa287–301 peptide. The data suggested that the enhanced immunogenicity of MAPs was not due to augmented Ag presentation or T cell stimulation. However, MAP-, but not monomer peptide-, containing immune complexes were potent substrates for Ab-dependent fixation of complement. These results demonstrate that the form of Ag responsible for inducing autoimmunity can profoundly influence the nature and magnitude of the immune response. Thus, molecular mimicry of tolerogenic and nontolerogenic self determinants might trigger autoimmunity under conditions of altered valence.
Cutting Edge: 4-1BB Is a Bona Fide CD8 T Cell Survival Signal
Authors: Takahashi Chikara , Mittler Robert S. , Vella Anthony T.
Published: December 2022

After recognition of Ag/MHC and ligation of a costimulatory molecule, resting T cells will clonally expand and then delete to very low levels. Previously, it was shown that deletion can be prevented by coinjection of cytokines or proinflammatory agents such as adjuvants. Here, we demonstrate that ligation of 4-1BB blocks deletion of superantigen-activated T cells in the absence of adjuvant or additional cytokine treatment. Nearly 10 times as many staphylococcal enterotoxin A-specific T cells were detected in the spleens of mice injected 21 days previously with staphylococcal enterotoxin A and an agonist anti-4-1BB Ab compared with mice given staphylococcal enterotoxin A and a control IgG. Even though both CD4- and CD8-activated T cells expressed 4-1BB, a higher proportion of CD8 T cells were rescued compared CD4 T cells. These data suggest that although 4-1BB provides costimulation, it may also promote long-term T cell survival.
Cutting Edge: An Inducible Sialidase Regulates the Hyaluronic Acid Binding Ability of CD44-Bearing Human Monocytes
Authors: Katoh Shigeki , Miyagi Taeko , Taniguchi Haruko , Matsubara Yu-ichi , Kadota Jun-ichi , Tominaga Akira , Kincade Paul W. , Matsukura Shigeru , Kohno Shigeru
Published: December 2022

Previous studies established that variable degrees and types of glycosylation can account for differences in the ability of CD44 to function as a receptor for hyaluronic acid. We have now used neuraminidase treatment to conclude that sialylation negatively regulates CD44 on the human monocytic cell line THP-1 and peripheral blood monocytes. Both of these cell types displayed increased receptor activity after overnight culture with LPS. Of particular interest, the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid completely blocked the LPS induced recognition of hyaluronic acid by THP-1 cells. Furthermore, acquisition of this characteristic paralleled induction of one type of sialidase activity. Monocytes may be capable of enzymaticly remodeling cell surface CD44, altering their ability to interact with the extracellular matrix.
An Essential Role for NF-κB in IL-18-Induced IFN-γ Expression in KG-1 Cells
Authors: Kojima Hirotada , Aizawa Yasushi , Yanai Yoshiaki , Nagaoka Katsue , Takeuchi Makoto , Ohta Tsunetaka , Ikegami Hakuo , Ikeda Masao , Kurimoto Masashi
Published: December 2022

IL-18 is a multifunctional cytokine playing various regulatory roles in the immune system including induced cytokine production. As a part of our ongoing studies on the molecular mechanisms of IL-18-induced IFN-γ production, we have examined the transcriptional regulation of the IFN-γ gene by IL-18 in a human myelomonocytic cell line, KG-1. On the basis of DNA/protein binding, we have determined an IL-18-inducible NF-κB binding site located at −786 to −776 of the IFN-γ gene regulatory region (designated KBBsite). Transient transfection of promoter-reporter gene constructs revealed that the KBBsite is required for full IL-18-induced activation of the IFN-γ gene transcription induced by IL-18. In addition, stable transformants of a dominant-negative form of the IκBα showed an inhibition of IL-18-dependent IκBα degradation, NF-κB activation, and expression of IFN-γ. These results are the first to show the actual significance of the NF-κB pathway in the regulation of IFN-γ gene expression by IL-18.
Thymic Shared Antigen-2: A Novel Cell Surface Marker Associated with T Cell Differentiation and Activation
Authors: Berzins Stuart P. , Davey Gayle M. , Randle-Barrett Elise S. , Malin Mark A. , Classon Brendan J. , Fraser Stuart , Boyd Richard L.
Published: December 2022

Thymic shared Ag-2 (TSA-2) is a 28-kDa, glycophosphatidylinitosol-linked cell surface molecule expressed on various T cell and thymic stromal cell subsets. It is expressed on most CD3−CD4−CD8−, CD4+CD8+, and CD3highCD4−CD8+ thymocytes but is down-regulated on ∼40% of CD3highCD4+CD8− thymocytes. Expression on peripheral TCR-αβ+ T cells is similar to that of CD3+ thymocytes, although a transient down-regulation occurs with cell activation. Consistent with the recent hypothesis that emigration from the thymus is an active process, recent thymic emigrants are primarily TSA-2−/low. TSA-2 expression reveals heterogeneity among subpopulations of CD3highCD4+CD8− thymocytes and TCR-γδ+ T cell previously regarded as homogenous. The functional importance of TSA-2 was illustrated by the severe block in T cell differentiation caused by adding purified anti-TSA-2 mAb to reconstituted fetal thymic organ culture. While each CD25/CD44-defined triple-negative subset was present, differentiation beyond the TN stage was essentially absent, and cell numbers of all subsets were significantly below those of control cultures. Cross-linking TSA-2 on thymocytes caused a significant Ca2+ influx but no increase in apoptosis, unless anti-TSA-2 was used in conjunction with suboptimal anti-CD3 mAb. Similar treatment of mature TSA-2+ T cells had no effect on cell survival or proliferation. This study reveals TSA-2 to be a functionally important molecule in T cell development and a novel indicator of heterogeneity among a variety of developing and mature T cell populations.
Emergence of CTL Coincides with Clearance of Virus During Primary Simian Immunodeficiency Virus Infection in Rhesus Monkeys
Authors: Kuroda Marcelo J. , Schmitz Jörn E. , Charini William A. , Nickerson Christine E. , Lifton Michelle A. , Lord Carol I. , Forman Meryl A. , Letvin Norman L.
Published: December 2022

The CTL response was characterized during primary SIV/macaque (SIVmac) infection of rhesus monkeys to assess its role in containing early viral replication using both an epitope-specific functional and an MHC class I/peptide tetramer-binding assay. The rapid expansion of a single dominant viral epitope-specific CTL population to 1.3–8.3% of circulating CD8+ peripheral blood and 0.3–1.3% of lymph node CD8+ T cells was observed, peaking at day 13 following infection. A subsequent decrease in number of these cells was then demonstrated. Interestingly, the percent of tetramer-binding CD8+ T cells detected in the lymph nodes of all evaluated animals was smaller than the percent detected in PBL. These epitope-specific CD8+ T cells expressed cell surface molecules associated with memory and activation. Early clearance of SIVmac occurred coincident with the emergence of the CTL response, suggesting that CTL may be important in containing virus replication. A higher percent of annexin V-binding cells was detected in the tetramer+ CD8+ T cells (range, from 33% to 75%) than in the remaining CD8+ T cells (range, from 3.3% to 15%) at the time of maximum CTL expansion in all evaluated animals. This finding indicates that the decrease of CTL occurred as a result of the death of these cells rather than their anatomic redistribution. These studies provide strong evidence for the importance of CTL in containing AIDS virus replication.
Differential Homing Commitments of Antigen-Specific T Cells After Oral or Parenteral Immunization in Humans
Authors: Kantele Anu , Zivny Jan , Häkkinen Miikka , Elson Charles O. , Mestecky Jiri
Published: December 2022

Animal experiments show that lymphocytes activated in the intestine circulate through mesenteric lymph nodes, lymphatics, and blood, returning to the gut. Homing into intestinal lamina propria is mediated by lymphocyte surface homing receptors, mainly the α4β7-integrin. We studied in humans whether intestinal T cells entering the blood upon antigenic activation would exhibit homing commitments to the gut. Volunteers were immunized with keyhole limpet hemocyanin (KLH) first orally and then parenterally or only parenterally, and the expression of α4β7 on T cells specific for KLH or tetanus toxoid was studied. Circulating T cells were depleted of α4β7+ cells by immunomagnetic selection. This depletion removed a significant proportion of the KLH-specific cells (mean decrease in proliferative response of 71%) in the orally immunized volunteers. No difference in the KLH-induced proliferation was found between the total and the α4β7-depleted populations in volunteers parenterally immunized with KLH, regardless of whether a preceding mucosal priming had taken place or not. In both immunization groups, the depletion of α4β7+ cells had no influence on the proliferative response to tetanus toxoid. We conclude that, in contrast to T cells activated by parenteral immunization, gut-derived T cells have preferential homing commitments to the gut. This commitment was no longer observed after a subsequent parenteral Ag administration. Besides showing that the site of Ag encounter determines the expression of homing receptors, the present study is the first to provide evidence for a circulation of newly activated Ag-specific intestinal T cells back to the gut in humans.
Cell-Surface Expression and Alloantigenic Function of a Human Nonclassical Class I Molecule (HLA-E) in Transgenic Mice
Authors: Pacasova Rita , Martinozzi Silvia , Boulouis Henri-Jean , Ulbrecht Matthias , Vieville Jean-Claude , Sigaux François , Weiss Elisabeth H. , Pla Marika
Published: December 2022

We have introduced the gene (E*01033) encoding the heavy chain of the human nonclassical MHC class I Ag, HLA-E, into the mouse genome. Two founder mice carry a 21-kb fragment, the others bear an 8-kb fragment. Each of the founder mice was mated to mice of an already established C57BL/10 transgenic line expressing human β2-microglobulin (β2m). Cell surface HLA-E was detected on lymph node cells by flow cytometry only in the presence of endogenous human β2m. However, HLA-E-reactive mouse CTL (H-2-unrestricted) lysed efficiently the target cells originating from HLA-E transgenic mice without human β2m, showing that the HLA-E protein can be transported to the cell surface in the absence of human β2m, presumably by association with murine β2m. Rejection of skin grafts from HLA-E transgenic mice demonstrates that HLA-E behaves as a transplantation Ag in mice. HLA-E transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal and human classical class I (HLA-B27) transgenic mice. Furthermore, results from split-well analysis indicate that the majority of the primary in vivo-induced CTL recognizes HLA-E as an intact molecule (H-2-unrestricted recognition) and not as an HLA-E-derived peptide presented by a mouse MHC molecule, although a small fraction (ranging from 4 to 21%) of the primary in vivo-induced CTL is able to recognize HLA-E in an H-2-restricted manner. Based on these observations, we conclude that HLA-E exhibits alloantigenic properties that are indistinguishable from classical HLA class I molecules when expressed in transgenic mice.
Induction of Primary Human CD8+ T Lymphocyte Responses In Vitro Using Dendritic Cells
Authors: Zarling Angela L. , Johnson Julia G. , Hoffman Robert W. , Lee David R.
Published: December 2022

The ability of two different human professional APCs, specifically macrophages (Mφ) and dendritic cells (DC), to stimulate primary responses in human CD8+ T lymphocytes was examined using both allogeneic and Ag-pulsed autologous APCs. CTL responses in CD8+ T lymphocytes isolated from HIV-uninfected donors were evaluated against six different HIV epitopes that are restricted by four different HLA alleles using autologous human PBMC-derived Mφ and DCs for primary stimulation. In a side-by-side experiment, immature DCs, but not Mφ, were able to prime a CTL response against the B14-restricted p24gag 298–306 epitope; mature DCs were also able to prime a response against this epitope. In addition, DCs were capable of priming CD8+ CTL responses against the B8-restricted p24gag 259–267 epitope. In contrast, Mφ were unable to prime strong CTL responses against other epitopes. Since the Ag-specific cytotoxic responses required subsequent rounds of restimulation before they could be detected, the ability of the allogeneic Mφ and DCs to directly prime CD8+ T lymphocyte responses without subsequent restimulation was examined. Similar to the aforementioned peptide-specific results, DCs were more efficient than Mφ in priming both allogeneic proliferative and cytotoxic responses in human CD8+ T lymphocytes. Collectively, these results promote an enhanced status for DCs in the primary stimulation of human CD8+ T lymphocytes.
Synergism for Cytokine-Mediated Disease During Concurrent Endotoxin and Viral Challenges: Roles for NK and T Cell IFN-γ Production
Authors: Nguyen Khuong B. , Biron Christine A.
Published: December 2022

Viral infections in humans or mice can result in increased sensitivity to challenges with bacteria, bacterial products, or cytokine administration. During lymphocytic choriomeningitis virus infections, mice are more sensitive to the lethal effects of bacterial endotoxin LPS, and in the experiments reported here, were observed at up to 10-fold lower doses in infected than in uninfected mice. The mechanisms responsible for heightened susceptibility under these conditions were evaluated. Kinetic studies demonstrated that virus-infected mice had 3- to 50-fold increases over uninfected mice in peak serum TNF, IL-12, and IFN-γ levels after LPS administration. All three cytokines contributed to lethality during dual challenge, because neutralization of any one of the factors protected from death. Production of TNF was not dependent on either NK or T cells. In contrast, these populations were the predominant sources of IFN-γ, as determined by lack of detectable IFN-γ production in NK and T cell-deficient mice and by intracellular cytokine expression in the cell subsets. Concordant with the demonstrations that both cell populations produced IFN-γ and that this factor was critical for lethality, removal of either subset alone was not sufficient to protect mice from death resulting from dual challenges. Increased resistance required absence of both cell subsets. Taken together, the data show that during viral infections, the normally protective immune responses can profoundly modify reactions to secondary heterologous challenges, to result in dysregulated cytokine expression and consequent heightened detrimental effects.
Mice with Th2-Biased Immune Systems Accept Orthotopic Corneal Allografts Placed in “High Risk” Eyes
Authors: Yamada Jun , Yoshida Munenori , Taylor Andrew W. , Streilein J. Wayne
Published: December 2022

CD4+ T cells of the Th1 type play a central role in acute rejection of solid tissue grafts, including orthotopic corneal allografts. Th1 cells, which mediate delayed hypersensitivity, are the polar opposites of CD4+ Th2 cells, and the latter cells cross-regulate Th1 cells through the unique pattern of cytokines they secrete. As such, Th2 cells may have a useful role to play in preventing rejection of corneal allografts. To test this possibility, the immune systems of adult mice were biased toward Th2 responses by immunization with keyhole limpet hemocyanin plus IFA. When immunized subsequently with either OVA or allogeneic corneal tissue, these mice acquired Ag-specific primed T cells of the Th2 type. More important, allogeneic corneas grafted into neovascularized eyes of Th2-biased mice experienced significantly enhanced survival. To demonstrate that enhanced survival was promoted by donor-specific Th2 cells, lymphoid cells from keyhole limpet hemocyanin-immune mice bearing healthy corneal allografts suppressed orthotopic corneal allograft rejection when adoptively transferred into naive, syngeneic recipients. We conclude that acceptance of corneal allografts in neovascularized mouse eyes can be significantly enhanced by biasing the recipient immune system toward Th2 responses.
Expression of L-Selectin Ligands by Transformed Endothelial Cells Enhances T Cell-Mediated Rejection
Authors: Biancone Luigi , Stamenkovic Ivan , Cantaluppi Vincenzo , Boccellino Mariarosaria , De Martino Antonella , Bussolino Federico , Camussi Giovanni
Published: December 2022

Recent immunohistochemical studies have suggested that L-selectin ligands may be implicated in the infiltration of tumors and rejected transplants by lymphocytes. In the present study, polyoma-middle T Ag-transformed endothelial cells (H.end), which typically form in vivo immunogenic vascular tumors resembling Kaposi’s sarcoma, were engineered to express L-selectin ligands by stable transfection with a cDNA encoding α(1,3/4)-fucosyltransferase (H.endft). The ability of these cells to form tumors in the s.c. tissues of normal and immunocompromised mice was then compared with that of H.end cells transfected with the hygromycin-resistance vector only (H.endhygro). H.endhygro cells rapidly formed local and metastatic tumors in normal syngeneic mice, leading to death within 2–3 mo postinjection. By contrast, tumors derived from H.endft cells displayed a slower rate of growth, an absence of metastasis, and marked lymphocyte infiltration. Animals bearing these tumors survived for a significantly longer duration than animals injected with H.endhygro cells. Alternatively, H.endft and H.endhygro cells formed tumors with comparable aggressiveness in immunocompromised mice, resulting in animal death within 3 wk of injection. H.endft but not H.endhygro cells supported L-selectin-dependent adhesion and cytolytic T cell activity in vitro. Taken together, our observations indicate that the in situ expression of fucosyltransferase may significantly influence the cellular immune response in endothelioma tumors. These results may be relevant in understanding the development of vascular opportunistic tumors such as Kaposi’s sarcoma.
Thymus and Autoimmunity: Production of CD25+CD4+ Naturally Anergic and Suppressive T Cells as a Key Function of the Thymus in Maintaining Immunologic Self-Tolerance
Authors: Itoh Misako , Takahashi Takeshi , Sakaguchi Noriko , Kuniyasu Yuhshi , Shimizu Jun , Otsuka Fujio , Sakaguchi Shimon
Published: December 2022

This study shows that the normal thymus produces immunoregulatory CD25+4+8− thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8− thymocytes, which constitute ∼5% of steroid-resistant mature CD4+8− thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8− thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8− or CD4−8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8− thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25−4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8− thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8− thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous α-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8− thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.
Multiple Roles for IL-12 in a Model of Acute Septic Peritonitis
Authors: Steinhauser Matthew L. , Hogaboam Cory M. , Lukacs Nickolas W. , Strieter Robert M. , Kunkel Steven L.
Published: December 2022

The present study addressed the role of IL-12 in a murine model of septic peritonitis, induced by cecal ligation and puncture (CLP). Although CLP surgery induced IL-12 production at 6 and 24 h after surgery, IL-12 immunoneutralization was clearly deleterious in this model: 54% of CLP mice receiving preimmune serum survived, whereas mice administered IL-12 antisera prior to CLP experienced a 25% survival rate. IL-12 immunoneutralization not only led to increased mortality, but also appeared to promote a shift away from IL-12 and IFN-γ, in favor of IL-10. This cytokine shift corresponded to changes in bacterial load, as CLP mice receiving IL-12 antiserum yielded more CFUs from the peritoneal cavity at 24 h after CLP. To address the role of bacterial infection in IL-12 antiserum-induced mortality following CLP, antibiotics were administered for 4 days after surgery. Despite regular antibiotic administration, IL-12 immunoneutralization still reduced survival in CLP mice. Furthermore, histology of the ceca revealed that mice administered IL-12 antisera failed to show typical organization of the damaged cecum wall. Accordingly, Gram staining revealed bacteria within peritoneal fluids from these mice, while peritoneal fluids from CLP mice that received preimmune serum and antibiotics were free of bacteria. Altogether, these data suggested multiple important roles for IL-12 in the evolution of murine septic peritonitis.
Thrombin-Induced p65 Homodimer Binding to Downstream NF-κB Site of the Promoter Mediates Endothelial ICAM-1 Expression and Neutrophil Adhesion
Authors: Rahman Arshad , Anwar Khandaker N. , True Andrea L. , Malik Asrar B.
Published: December 2022

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-κB site (−533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-κB site (−223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-κB site is critical for thrombin inducibility. NF-κB-directed luciferase activity increased ∼3-fold when cells transfected with the plasmid pNF-κBLUC containing five copies of consensus NF-κB site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-κB was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-κBp65 (Rel A) to downstream NF-κB site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin’s action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-κBp65 binding to the downstream NF-κB site of ICAM-1 promoter after proteolytically activated receptor-1 activation.
Membrane-Anchored Forms of Lipopolysaccharide (LPS)-Binding Protein Do Not Mediate Cellular Responses to LPS Independently of CD14
Authors: Tapping Richard I. , Orr Sally L. , Lawson Evangeline M. , Soldau Katrin , Tobias Peter S.
Published: December 2022

Inflammatory responses of myeloid cells to LPS are mediated through CD14, a glycosylphosphatidylinositol-anchored receptor that binds LPS. Since CD14 does not traverse the plasma membrane and alternatively anchored forms of CD14 still enable LPS-induced cellular activation, the precise role of CD14 in mediating these responses remains unknown. To address this, we created a transmembrane and a glycosylphosphatidylinositol-anchored form of LPS-binding protein (LBP), a component of serum that binds and transfers LPS to other molecules. Stably transfected Chinese hamster ovary (CHO) fibroblast and U373 astrocytoma cell lines expressing membrane-anchored LBP (mLBP), as well as separate CHO and U373 cell lines expressing membrane CD14 (mCD14), were subsequently generated. Under serum-free conditions, CHO and U373 cells expressing mCD14 responded to as little as 0.1 ng/ml of LPS, as measured by NF-κB activation as well as ICAM and IL-6 production. Conversely, the vector control and mLBP-expressing cell lines did not respond under serum-free conditions even in the presence of more than 100 ng/ml of LPS. All the cell lines exhibited responses to less than 1 ng/ml of LPS in the presence of the soluble form of CD14, demonstrating that they are still capable of LPS-induced activation. Taken together, these results demonstrate that mLBP, a protein that brings LPS to the cell surface, does not mediate cellular responses to LPS independently of CD14. These findings suggest that CD14 performs a more specific role in mediating responses to LPS than that of simply bringing LPS to the cell surface.
Detection of Functional Platelet-Activating Factor Receptors on Human Tonsillar B Lymphocytes
Authors: Bastien Yolande , Toledano Baruch J. , Mehio Noha , Cameron Lisa , Lamoukhaid Bouchaib , Renzi Poalo , Hamid Qutayba , Mazer Bruce D.
Published: December 2022

Although platelet-activating factor (PAF) receptors have been found on B lymphoblastoid cell lines, the action of PAF on freshly isolated human B cells has not been clearly demonstrated. Using a sensitive semiquantitative reverse-transcriptase PCR, we have found PAF receptor mRNA expressed by tonsillar B lymphocytes, but little in T lymphocytes. Examination of Percoll-fractionated tonsillar B cells indicated that the low density (primarily germinal center cells) and medium density fractions had approximately twofold more PAF receptor mRNA relative to the high density fraction. PAF (10−7 M) stimulated increases in intracellular Ca2+ that were consistently higher in the low and medium density B lymphocytes compared with high density cells. The PAF receptor antagonist Web 2170 inhibited this. Addition of PAF, but not lyso- or enantio-PAF, induced four- to sixfold greater synthesis of IgM and IgG in low and medium density cells compared with unstimulated controls, but had little effect on Ig production by high density cells. To investigate how PAF may influence Ig synthesis, PAF-stimulated B cells were examined for production of the Th2-type cytokines IL-4 and IL-13. PAF induced IL-4 and IL-13 mRNA expression in 17% of CD20+ cells, and IL-4 was detected in cell supernatants after 48–72 h of culture. Together, these data strongly suggest that functional PAF receptors are expressed on B cells in tonsils.
Endothelial Expression of VCAM-1 in Experimental Crescentic Nephritis and Effect of Antibodies to Very Late Antigen-4 or VCAM-1 on Glomerular Injury
Authors: Allen Andrew R. , McHale Julie , Smith Jennifer , Cook H. Terence , Karkar Ayman , Haskard Dorian O. , Lobb Roy R. , Pusey Charles D.
Published: December 2022

The migration of leukocytes into glomeruli in crescentic glomerulonephritis is fundamental to pathogenesis, and offers important therapeutic opportunities. We addressed the importance of VCAM-1, and its leukocyte ligand very late antigen-4 (VLA-4), in such leukocyte migration. In a rat model of nephrotoxic nephritis, glomerular expression of VCAM-1, studied by immunohistochemistry, was up-regulated by day 6 of nephritis. To quantify kidney endothelial VCAM-1 expression, a differential radiolabeled mAb technique was used, which demonstrated that protein expression was not up-regulated by day 2 of nephritis, but rose threefold between days 2 and 5, and remained elevated until at least day 28. An in vivo study was then performed, using blocking mAbs to either VCAM-1 or VLA-4, starting mAb treatment on the day prior to disease induction, and continuing until animals were sacrificed at day 7. mAbs to VLA-4 significantly attenuated renal injury (albuminuria, glomerular fibrinoid necrosis, and crescent formation), but mAbs to VCAM-1 had no significant effect. Surprisingly, the number of leukocytes within glomeruli was unaffected by anti-VLA-4 mAb therapy, despite the reduction in renal injury. Paradoxically, classical markers of macrophage activation were increased in the anti-VLA-4- and anti-VCAM-1-treated animals. This study demonstrates that kidney endothelial VCAM-1, in contrast to ICAM-1, is not up-regulated by day 2 of nephrotoxic nephritis, and plays little part in early leukocyte influx into glomeruli. However, VLA-4 is an important mediator of glomerular injury, operating after transendothelial leukocyte migration, and presumably binding to alternate ligands within the kidney.
Organ-Specific (Localized) Synthesis of Ig Light Chain Amyloid
Authors: Hamidi Asl Kamran , Liepnieks Juris J. , Nakamura Masaaki , Benson Merrill D.
Published: December 2022

Ig amyloidosis is usually a systemic disease with multisystem involvement. However, in a significant number of cases amyloid deposition is limited to one specific organ. It has not been determined if the Ig light chain (LC) amyloid precursor protein in localized amyloidosis is synthesized by circulating plasma cells with targeting of the amyloid fibril-forming process to one specific organ, or whether the synthesis of Ig LC and fibril formation occurs entirely as a localized process. In the present study local synthesis of an amyloid fibril precursor LC was investigated. Amyloid fibrils were isolated from a ureter that was obstructed by extensive infiltration of the wall with amyloid. Amino acid sequence analysis of the isolated fibril subunit protein proved it to be derived from a λII Ig LC. Plasma cells within the lesion stained positively with labeled anti-λ Ab and by in situ hybridization using an oligonucleotide probe specific for λ-LC mRNA. RT-PCR of mRNA extracted from the tumor and direct DNA sequencing gave the nucleotide sequence coding specifically for the λII amyloid subunit protein, thus confirming local synthesis of the LC protein.
Impaired Allostimulatory Capacity of Peripheral Blood Dendritic Cells Recovered from Hepatitis C Virus-Infected Individuals
Authors: Kanto Tatsuya , Hayashi Norio , Takehara Tetsuo , Tatsumi Tomohide , Kuzushita Noriyoshi , Ito Akihiko , Sasaki Yutaka , Kasahara Akinori , Hori Masatsugu
Published: December 2022

In hepatitis C virus (HCV) infection, Th responses are implicated in the pathogenesis of liver disease. The dendritic cell (DC) is the most potent activator of CD4 T cells for supporting Th1 differentiation. To clarify the roles of DC of HCV-infected individuals in the development of CD4 T cell responses, we generated peripheral DC with GM-CSF and IL-4 from 24 chronic hepatitis C patients and 14 healthy donors. We then compared their potentials for stimulating allogeneic CD4 T cells, autologous CD4 T cells against influenza A or HCV core Ags, and cytokine production. The DC from the patients (HCV-DC) expressed lower degrees of CD86 than DC from the donors (N-DC), whereas no difference was found in the HLA molecules and other costimulators. HCV-DC stimulated allogeneic T cells less than N-DC; however, influenza A- or core-pulsed HCV-DC retained the potentials for autologous T cell proliferation. In allogeneic DC/T cell cultures, the IFN-γ levels with HCV-DC were lower than those with N-DC, which may be related to the low expressions of IL-12 p35 and p40 transcripts in HCV-DC. The stimulation with LPS disclosed that HCV-DC is less potent in IL-12 p70 production than N-DC. In the autologous cultures, the pulsing of the Ags to HCV-DC increased the IL-12 p40 and IFN-γ production and up-regulated the transcription of both IL-12 subunits. Exogenous IL-2 or IL-12 restored the low allogeneic T cell proliferation with HCV-DC in a dose-dependent manner. Therefore, low expression of CD86 and/or IL-12 is crucially involved in the low allostimulatory capacity of HCV-DC. Low IL-12 and low IFN-γ milieu with HCV-DC on encounters with alloantigens may impede Th1 polarization.
Defective Self-Reactive Antibody Repertoire of Serum IgM in Patients with Hyper-IgM Syndrome
Authors: Lacroix-Desmazes Sébastien , Resnick Igor , Stahl Dorothea , Mouthon Luc , Espanol Teresa , Levy Jacov , Kaveri Srini V. , Notarangelo Luigi , Eibl Martha , Fischer Alain , Ochs Hans , Kazatchkine Michel D.
Published: December 2022

We have analyzed the self-reactive repertoires of IgM and IgG Abs in the serum of 19 patients with hyper-IgM syndrome (HIM) by means of a quantitative immunoblotting technique that allows for a quantitative comparison of Ab repertoires in health and disease by multiparametric statistical analysis. Normal tissue extracts of liver, lung, stomach, and kidney were used as sources of self Ags. Extracts of Pseudomonas aeruginosa and Staphylococcus epidermidis were used as sources of nonself Ags. We demonstrate a significant bias in repertoires of reactivities of IgM of patients with HIM with self Ags. Ab repertoires of IgM toward nonself Ags did not differ, however, between patients and controls. No difference was found between IgM repertoires of untreated patients and those of patients receiving substitutive treatment with i.v. IgG. IgG in the serum of HIM patients lacked reactivity with self Ags, although it exhibited a pattern of reactivity with nonself Ags that was similar to that of IgG of healthy controls. The data demonstrate that functional CD40-CD40 ligand interactions are essential for the selection of natural self-reactive B cell repertoires.
Diminished Levels of Protein Kinase A RIα and RIβ Transcripts and Proteins in Systemic Lupus Erythematosus T Lymphocytes
Authors: Laxminarayana Dama , Khan Islam U. , Mishra Nilamadhab , Olorenshaw Irene , Taskén Kjetil , Kammer Gary M.
Published: December 2022

Deficient type I protein kinase A phosphotransferase activity occurs in the T cells of 80% of subjects with systemic lupus erythematosus (SLE). To investigate the mechanism of this deficient isozyme activity, we hypothesized that reduced amounts of type I regulatory (RI) isoform transcripts, RIα and RIβ, may be associated with a diminution of RIα and/or RIβ protein. Sixteen SLE subjects with a mean (±1 SD) SLE disease activity index of 12.4 ± 7.2 were studied. Controls included 16 normal subjects, six subjects with primary Sjögren’s syndrome (SS), and three subjects with SS/SLE overlap. RT-PCR revealed that normal, SS, SS/SLE, and SLE T cells expressed mRNAs for all seven R and catalytic (C) subunit isoforms. Quantification of mRNAs by competitive PCR revealed that the ratio of RIα mRNA to RIβ mRNA in normal T cells was 3.4:1. In SLE T cells there were 20 and 49% decreases in RIα and RIβ mRNAs (RIβ; p = 0.008), respectively, resulting in an RIα:RIβ mRNA of 5.3:1. SS/SLE T cells showed a 72.5% decrease in RIβ mRNA compared with normal controls (p = 0.01). Immunoblotting of normal T cell RIα and RIβ proteins revealed a ratio of RIα:RIβ of 3.2:1. In SLE T cells, there was a 30% decrease in RIα protein (p = 0.002) and a 65% decrease in RIβ protein (p < 0.001), shifting the ratio of RIα:RIβ protein to 6.5:1. T cells from 25% of SLE subjects lacked any detectable RIβ protein. Analysis of several lupus T cell lines demonstrated a persistent deficiency of both proteins, excluding a potential effect of disease activity. In conclusion, reduced expression of RIα and RIβ transcripts is associated with a decrement in RIα and RIβ proteins and may contribute to deficient type I protein kinase A isozyme activity in SLE T cells.
Cutting Edge: A Small Molecule Antagonist of LFA-1-Mediated Cell Adhesion
Authors: Kelly Terence A. , Jeanfavre Deborah D. , McNeil Daniel W. , Woska Joseph R. , Reilly Patricia L. , Mainolfi Elizabeth A. , Kishimoto Karen M. , Nabozny Gerald H. , Zinter Rosemarie , Bormann Barbara-Jean , Rothlein Robert
Published: December 2022

LFA-1 (CD18,CD11a) is a cell-adhesion molecule that mediates critical immunological processes. In this paper we report the discovery and characterization of (R)-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (BIRT 377), an orally bioavailable small molecule that interacts specifically with LFA-1 via noncovalent binding to the CD11a chain and prevents LFA-1 from binding to its ligand, ICAM-1. BIRT 377 inhibits lymphocyte activity both in vitro and in vivo, in functional assays that require LFA-1-mediated cell adhesion. These results demonstrate that LFA-1-mediated leukocyte adhesion can be antagonized with noncharged, low m.w. molecules and suggest that the potential therapeutic value of adhesion inhibitors can be attained with a small, orally bioavailable compound.
Roles of Intracellular Calcium and NF-κB in theClostridium difficileToxin A-Induced Up-Regulation and Secretion of IL-8 from Human Monocytes
Authors: Jefferson Kimberly K. , Smith Michael F. , Bobak David A.
Published: December 2022

Clostridium difficile causes an intense inflammatory colitis through the actions of two large exotoxins, toxin A and toxin B. IL-8 is believed to play an important role in the pathophysiology of C. difficile-mediated colitis, although the mechanism whereby the toxins up-regulate the release of IL-8 from target cells is not well understood. In this study, we investigated the mechanisms through which toxin A induces IL-8 secretion in human monocytes. We found that cellular uptake of toxin A is required for the up-regulation of IL-8, an effect that is not duplicated by a recombinant toxin fragment comprising the cell-binding domain alone. Toxin A induced IL-8 expression at the level of gene transcription and this effect occurred through a mechanism requiring intracellular calcium and calmodulin activation. Additionally, the effects of toxin A were inhibited by the protein tyrosine kinase inhibitor genistein, but were unaffected by inhibitors of protein kinase C and phosphatidylinositol-3 kinase. We determined that toxin A activates nuclear translocation of the transcription factors NF-κB and AP-1, but not NF-IL-6. NF-κB inhibitors blocked the ability of toxin A to induce IL-8 secretion, and supershift analysis indicated that the major isoform of NF-κB activated by the toxin is a p50-p65 heterodimer. This study is the first to identify intracellular signaling pathways and transcription factors involved in the C. difficile toxin-mediated up-regulation of IL-8 synthesis and release by target cells. This information should increase our understanding of the pathogenesis of C. difficile colitis and the nature of IL-8 gene regulation as well.
Activation of Caspases in Lethal Experimental Hepatitis and Prevention by Acute Phase Proteins
Authors: Van Molle Wim , Denecker Geertrui , Rodriguez Ivan , Brouckaert Peter , Vandenabeele Peter , Libert Claude
Published: December 2022

Lethal hepatitis can be induced by an agonistic anti-Fas Ab in normal mice or by TNF in mice sensitized to d-(+)-galactosamine or actinomycin D. In all three models, we found that apoptosis of hepatocytes is an early and necessary step to cause lethality. In the three models, we observed activation of the major executioner caspases-3 and -7. Two acute-phase proteins, α1-acid glycoprotein and α1-antitrypsin, differentially prevent lethality: α1-acid glycoprotein protects in both TNF models and not in the anti-Fas model, while α1-antitrypsin confers protection in the TNF/d-(+)-galactosamine model only. The protection is inversely correlated with activation of caspase-3 and caspase-7. The data suggest that activation of caspase-3 and -7 is essential in the in vivo induction of apoptosis leading to lethal hepatitis and that acute phase proteins are powerful inhibitors of apoptosis and caspase activation. Furthermore, Bcl-2 transgenic mice, expressing Bcl-2 specifically in hepatocytes, are protected against a lethal challenge with anti-Fas or with TNF/d-(+)-galactosamine, but not against TNF/actinomycin D. The acute-phase proteins might constitute an inducible anti-apoptotic protective system, which in pathology or disturbed homeostasis prevents excessive apoptosis.