bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody Binding to Individual Short Consensus Repeats of Decay-Accelerating Factor Enhances Enterovirus Cell Attachment and Infectivity
Authors: Shafren Darren R. , Dorahy Douglas J. , Thorne Rick F. , Kinoshita Taroh , Barry Richard D. , Burns Gordon F.
Published: January 2023

Decay-accelerating factor (DAF), a widely expressed membrane complement-regulatory protein, is utilized as a cellular receptor by many human enteric pathogens. We show here that the binding of two enteroviruses to individual short consensus repeats (SCR) of DAF on the cell surface is greatly augmented by mAb binding to an alternate SCR: Coxsackievirus A21 binding to the SCR1 of DAF is increased by Ab binding to SCR3 and, conversely, Echovirus 7 binding to SCR3 is enhanced severalfold by Ab binding to SCR1. These Ab-induced increases in viral binding also resulted in increased viral infectivity. Using purified soluble DAF in a solid phase assay it was found that Ab binding to SCR1 is increased greatly in the presence of an Ab against SCR3 and, reciprocally, Ab against SCR1 greatly increases Ab binding to SCR3. In contrast to the results obtained with the larger viral particles, however, this reciprocal Ab-induced enhancement of binding is not seen when measuring Ab binding to membrane-bound DAF SCR on the cell surface. These findings provide a possible explanation for functional differences between membrane-bound and soluble DAF with implications for a potential role for DAF-binding molecules in regulating DAF function. This is the first demonstration of enhancement of viral infectivity mediated by Ab against the viral receptor.
Distinct NFAT Family Proteins Are Involved in the Nuclear NFAT-DNA Binding Complexes from Human Thymocyte Subsets
Authors: Amasaki Yoshiharu , Masuda Esteban S. , Imamura Ryu , Arai Ken-ichi , Arai Naoko
Published: January 2023

The nuclear factor of activated T cells (NFAT) is involved in the transcriptional induction of cytokine and other immunoregulatory genes during an immune response. Among four distinct NFAT family members identified to date, mRNAs of NFAT1, NFATc, and NFATx are expressed in the thymus. Here, we report the distribution of these three NFAT family members in human fetal thymocyte subsets and in peripheral mature T cells. We show that NFATx mRNA was expressed in all T lymphocyte subsets tested and was highest in CD4+CD8+ double positive (DP) thymocytes. Conversely, NFAT1 mRNA was preferentially expressed in the mature CD4+ single positive (SP) populations. NFATc mRNA was present at low levels in all subsets but strongly induced upon treatment with phorbol ester and calcium ionophore. Interestingly, we detected NFAT-DNA binding complexes in DP thymocytes, albeit at lower levels than in CD4 SP cells. Corresponding to the mRNA expression, we observed that NFATx was responsible for the NFAT-DNA binding in DP thymocytes. Moreover, this DNA binding was inhibited by cyclosporin A, indicating that NFATx nuclear translocation was regulated by the calcineurin phosphatase in DP thymocytes. For the CD4 SP populations, NFAT1 and NFATc, and to some extent NFATx, were responsible for the NFAT-DNA binding complexes. These results indicate that NFAT family members are differentially regulated during the development of T cells, and that NFATx may play a distinct role in calcineurin-dependent signaling in DP thymocytes.
CKβ-11/Macrophage Inflammatory Protein-3β/EBI1-Ligand Chemokine Is an Efficacious Chemoattractant for T and B Cells
Authors: Kim Chang H. , Pelus Louis M. , White John R. , Applebaum Edward , Johanson Kyung , Broxmeyer Hal E.
Published: January 2023

We examined the functional properties of CKβ-11/MIP-3β/ELC, a recently reported CC chemokine that specifically binds to a chemokine receptor, EBI1/BLR2/CCR7. CKβ-11/MIP-3β/ELC is distantly related to other CC and CXC chemokines in primary amino acid sequence structure. Recombinant human CKβ-11/MIP-3β/ELC expressed from a mammalian cell system showed potent chemotactic activity for T cells and B cells but not for granulocytes and monocytes. An optimal concentration of CKβ-11/MIP-3β/ELC attracted most input T cells within 3 h, a chemotactic activity comparable with that of stromal cell derived factor 1 (SDF-1), a highly efficacious CXC chemokine. CKβ-11/MIP-3β/ELC equally attracted naive CD45RA+ and memory type CD45RO+ T cells. CKβ-11/MIP-3β/ELC also strongly attracted both CD4+ and CD8+ T cells, but the attraction for CD4+ T cells was greater. CKβ-11/MIP-3β/ELC was also a more efficacious chemoattractant for B cells than MIP-1α, a known B cell chemoattractant. CKβ-11/MIP-3β/ELC induced actin polymerization in lymphocytes, and chemotaxis was completely blocked by pertussis toxin showing its receptor, most likely EBI1/BLR2/CCR7, is coupled to a Gαi protein. CKβ-11/MIP-3β/ELC induced calcium mobilization in lymphocytes, which could be desensitized by SDF-1, suggesting possible cross-regulation in their signaling. Human CKβ-11/MIP-3β/ELC attracted murine splenocytes suggesting functional conservation of CKβ-11/MIP-3β/ELC between human and mouse. The efficacy of chemoattraction by CKβ-11/MIP-3β/ELC and tissue expression of its mRNA suggest that CKβ-11/MIP-3β/ELC may be important in trafficking of T cells in thymus, and T cell and B cell migration to secondary lymphoid organs.
HIV-1/Simian Immunodeficiency Virus Infection of Human and Nonhuman Primate Lymphocytes Results in the Migration of CD2+ T Cells into the Intestine of Engrafted SCID Mice
Authors: Donze Hollie Hale , Cummins James E. , Schwiebert Rebecca S. , Kantele Anu , Han Yuncheng , Fultz Patricia N. , Jackson Susan , Mestecky Jiri
Published: January 2023

Increased lymphocytic infiltration of intestinal tissues has been observed in patients infected with HIV-1 and in SIV-infected rhesus macaques. To determine whether HIV-1 and SIV infections influence the homing of human and nonhuman primate PBMC to intestinal tissues, we engrafted SCID mice with human or nonhuman primate PBMC and infected them with either cell-free or cell-associated HIV-1 or SIV. In mice that received both PBMC and virus, human or nonhuman primate CD2+ T cells were found in intestinal tissues, primarily in the intraepithelial lymphocyte compartment and lamina propria. Immunomagnetic sorting revealed that these cells were derived from the CD4+ population. Using gag-specific primers, PCR analysis of these tissues detected the presence of HIV-1 proviral DNA. However, in SCID mice that were engrafted with either human or nonhuman primate PBMC and no HIV-1 or SIV, CD2+ T cells were not detected in intestinal tissues. These results indicate that HIV-1 and SIV can modulate the migratory properties of human and nonhuman primate T cells in the SCID mouse model.
Cutting Edge: IL-12 and IL-18 Differentially Regulate the Transcriptional Activity of the Human IFN-γ Promoter in Primary CD4+ T Lymphocytes
Authors: Barbulescu Karina , Becker Christoph , Schlaak Jörg F. , Schmitt Edgar , Meyer zum Büschenfelde Karl-Hermann , Neurath Markus F.
Published: January 2023

We analyzed the molecular mechanisms by which IL-12 and IL-18 induce transcriptional activity of the IFN-γ promoter in primary human CD4+ T cells. In transfection experiments, we found that IL-18 directly induces IFN-γ promoter activity, whereas significant activation with IL-12 required costimulation with αCD3/CD28. Furthermore, IL-12 caused in vivo protection of a STAT4 (−236) binding site, whereas stimulation with IL-18 or IL-12 plus αCD3/CD28 induced occupancy of a downstream AP-1 site. Mutation of this AP-1 site abrogated both IL-12- and IL-18-mediated promoter activation, whereas mutation of the STAT site inhibited IL-12-dependent activation. These data suggest that both AP-1 and STAT4 are required for IL-12-dependent IFN-γ promoter activity, whereas IL-18 causes direct activation via AP-1. This differential activation of the IFN-γ promoter gives further insights into molecular pathways governing Th1 T cell development and differentiation.
CD8+ Cells Are Not Necessary for Allograft Rejection or the Induction of Apoptosis in an Experimental Model of Small Intestinal Transplantation
Authors: Krams Sheri M. , Hayashi Michihiro , Fox Christine K. , Villanueva Janeth C. , Whitmer Karen J. , Burns Washington , Esquivel Carlos O. , Martinez Olivia M.
Published: January 2023

Allospecific CTL can function as cellular effectors of solid organ graft rejection; however, the specific mechanisms of cell damage remain undetermined. In this study we examined the role of CD8+ T cells in apoptosis and rejection of small intestinal allografts. ACI rat intestinal grafts transplanted into Lewis rat recipients showed apoptosis of epithelial crypt cells on day 3 posttransplant as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining. By day 7 numerous apoptotic crypt cells were detected in allografts, but were rarely observed in FK506-treated allograft recipients, isografts, or native intestine of allograft recipients. To further investigate the mechanism of rejection, recipient rats were depleted of CD8+ cells by treatment with OX-8 mAbs the day before and the day after transplantation of rat small intestinal allografts. Depletion of CD8+ cells from allograft recipients did not alter the tempo or the histologic features of rejection compared with those in the control (IgG-treated) group. Moreover, there was no difference in the number of apoptotic crypt epithelial cells in the grafts of control and CD8-depleted rats. Reverse transcriptase-PCR analyses determined there were similar levels of transcripts for Fas, Fas ligand, perforin, and granzyme B in control and CD8-depleted allograft recipients. By Western blot it was determined that the levels of Fas ligand protein were increased in the CD8-depleted group compared with those in control and FK506-treated allograft recipients. These data suggest that CD8 cells are not required for tissue injury or apoptotic cell death in small intestine allograft rejection.
Phorbol Esters Induce Differentiation of Human CD34+ Hemopoietic Progenitors to Dendritic Cells: Evidence for Protein Kinase C-Mediated Signaling
Authors: Davis Thomas A. , Saini Abha A. , Blair Patrick J. , Levine Bruce L. , Craighead Nancy , Harlan David M. , June Carl H. , Lee Kelvin P.
Published: January 2023

The intracellular signals that mediate the differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have found that the phorbol ester PMA by itself induced 47% ± 8.7% of input human CD34+ hemopoietic progenitors to differentiate into cells with morphology and surface Ag phenotype characteristic of DC by day 7 of culture. Functionally, PMA-generated DC processed and presented whole soluble Ag and also induced resting T cell proliferation and Ag-specific CTL effector function. Unlike cytokine-driven DC differentiation, PMA suppressed proliferation and induced cell death (in part via apoptosis) in cells that did not differentiate to DC. The effects of PMA were blocked by inhibitors of protein kinase C activation, suggesting a central role for this signaling molecule. PMA-mediated signaling also induced expression of the RelB transcription factor, an NF-κB family member implicated in DC differentiation. These findings suggest that phorbol esters activate protein kinase C, which then initiates the terminal component of an intracellular signaling pathway(s) involved in the DC differentiation of CD34+ hemopoietic progenitors.
IFN-γ Receptor Deletion Prevents Autoantibody Production and Glomerulonephritis in Lupus-Prone (NZB × NZW)F1 Mice
Authors: Haas Cordula , Ryffel Bernhard , Le Hir Michel
Published: January 2023

(NZB × NZW)F1 female (BW) mice spontaneously develop an autoimmune disease, characterized by the production of autoantibodies (autoAbs) and glomerulonephritis, which can be delayed by neutralizing IFN-γ Abs and accelerated by IFN-γ injections. To define the role of IFN-γ in the pathogenesis of glomerulonephritis, we established a population of BW mice deficient in IFN-γR (BWγR−/−) by repeated crossing; these mice were compared with BWγR+/+ and +/− littermates. Of the BWγR+/+ and +/− mice, 50% showed immune complex glomerulonephritis with heavy proteinuria at 8 mo of age, while only 10% of the BWγR−/− mice were affected at 14 mo. The serum concentration of anti-dsDNA and anti-histone Abs was dramatically reduced in BWγR−/− mice. The role of IFN-γ in promoting class switch to IgG2a and IgG3 could not fully account for the impaired production of anti-dsDNA in BWγR−/− animals since, IgM and IgG1 levels were also reduced. There was a high incidence of B cell lymphoma in the BWγR−/− mice, which might be related to the suppression of autoAb production. Thus, the absence of glomerulonephritis in BWγR−/− mice is likely due to a dramatic yet unexplained effect of the inactivation of IFN-γ signaling on autoAb production.
CTLA-4 Regulates Tolerance Induction and T Cell Differentiation In Vivo
Authors: Walunas Theresa L. , Bluestone Jeffrey A.
Published: January 2023

Cytotoxic T lymphocyte Ag-4 (CTLA-4; CD152) is an important T cell regulatory molecule. In vitro experiments have shown that the blockade of signals through CTLA-4 augments T cell expansion, while CTLA-4 cross-linking results in decreased T cell proliferation due to decreased IL-2 production. However, less is known about the role of CTLA-4 in regulating an ongoing immune response. In this study, we examined the role of CTLA-4 in the expansion, decline, tolerization, and differentiation of T cells following treatment with staphylococcal enterotoxin B (SEB). Anti-CTLA-4 treatment resulted in increased numbers of SEB-reactive T cells and blockade of subsequent tolerance induction. Further examination of the SEB-reactive cells from anti-CTLA-4-treated mice demonstrated that both the CD4+ and CD8+ Vβ8+ T cells produced IL-4, providing evidence that not only do signals through CTLA-4 regulate T cell-tolerizing events, but they also play an important role in the differentiation of T cells in vivo.
Intraepithelial Infiltration by Mast Cells with Both Connective Tissue-Type and Mucosal-Type Characteristics in Gut, Trachea, and Kidneys of IL-9 Transgenic Mice
Authors: Godfraind Catherine , Louahed Jamila , Faulkner Helen , Vink Anne , Warnier Guy , Grencis Richard , Renauld Jean-Christophe
Published: January 2023

IL-9 transgenic mice were analyzed for the presence of mast cells in different tissues. In these mice, increased mast cell infiltration was found in the gastric and intestinal epithelium as well as in the upper airways and kidney epithelium, but not in other organs, such as skin. IL-9 transgenic mast cells do not show signs of massive degranulation such as that found in IL-4 transgenic mice and are not involved in spontaneous pathologic changes. Gastric mast cells showed a phenotype related to connective-type mast cells, since they were stained by safranin, and strong expression of mouse mast cell protease-4 and -5 was found in this organ. However, they also expressed proteases related to the mucosal cell type, such as mouse mast cell protease-1 and -2. In vitro, although IL-9 by itself did not induce mast cell development from bone marrow progenitors, it strongly synergized with stem cell factor for the growth and differentiation of mast cells expressing the same protease pattern as that observed in IL-9 transgenic mice. Since constitutive stem cell factor expression was observed in vivo, and anti-c-Kit Abs inhibited IL-9 transgenic mastocytosis in the gut, this synergistic combination of factors is likely to be responsible for the mastocytosis observed in IL-9 transgenic mice. Taken together, these data demonstrate that IL-9 induces the in vivo amplification of a nonclassical mast cell subset with a mucosal localization but expressing proteases characteristic of both connective tissue-type and mucosal mast cells.
Human Polymorphonuclear Leukocytes Adhere to Complement Factor H Through an Interaction That Involves αMβ2 (CD11b/CD18)
Authors: DiScipio Richard G. , Daffern Pamela J. , Schraufstätter Ingrid U. , Sriramarao Pragda
Published: January 2023

The work presented here demonstrates that human complement factor H is an adhesion ligand for human neutrophils but not for eosinophils. The adherence of polymorphonuclear leukocytes (PMNs) to plastic wells coated with factor H depended on divalent metal ions and was augmented by C5a and TNF-α. PMN adhesion to factor H in the presence or absence of C5a was blocked specifically by mAbs against CD11b or CD18. Affinity purification using factor H Sepharose followed by immunoprecipitation using mAbs to various integrin chains identified Mac-1 (CD11b/CD18) as a factor H binding receptor. The presence of surface bound factor H enhanced neutrophil activation resulting in a two- to fivefold increase in the generation of hydrogen peroxide by PMNs stimulated by C5a or TNF-α. When factor H was mixed with PMNs, 1.4 to 3.8-fold more cells adhered to immobilized heparin or chondroitin A. In addition, augmented adhesion of PMNs was measured when factor H, but not HSA or C9, was absorbed to wells that were first coated with heparin or chondroitin A. The adhesion of PMNs to glycosaminoglycan-factor H was blocked by mAbs to CD11b and CD18. These studies demonstrate that factor H is an adhesion molecule for human neutrophils and suggest that the interaction of factor H with glycosaminoglycans may facilitate the tethering of this protein in tissues allowing factor H to serve as a neutrophil adhesion ligand in vivo.
IFN-γ Limits Macrophage Expansion in MRL-FaslprAutoimmune Interstitial Nephritis: A Negative Regulatory Pathway
Authors: Schwarting Andreas , Moore Kathryn , Wada Takashi , Tesch Gregory , Yoon Hyung-Jin , Kelley Vicki Rubin
Published: January 2023

IFN-γ is capable of enhancing and limiting inflammation. Therefore, an increase in IFN-γ in autoimmune MRL-Faslpr mice could exacerbate or thwart renal injury. We have established a retroviral gene transfer approach to incite interstitial nephritis in MRL-Faslpr mice that is rapid, enduring, and circumscribed. Renal tubular epithelial cells (TEC) were genetically modified to secrete macrophage (Mφ) growth factors (CSF-1-TEC, GM-CSF-1-TEC) and infused under the renal capsule. To determine the impact of IFN-γ in Mφ growth factor-incited renal injury, we constructed a MRL-Faslpr IFN-γ-receptor (IFN-γR)-deficient strain. Gene transfer of CSF-1 or GM-CSF incited more severe interstitial nephritis in IFN-γR-deficient than in IFN-γR-intact MRL-Faslpr mice, consisting of an increase of Mφ. To determine the mechanism responsible for the increase in Mφ in IFN-γR-deficient MRL-Faslpr mice, we evaluated Mφ proliferation, apoptosis, and recruitment. Proliferation of bone marrow Mφ from IFN-γR-intact MRL-Faslpr costimulated with CSF-1 or GM-CSF and IFN-γ was reduced twofold, while the IFN-γR-deficient MRL-Faslpr bone marrow Mφ remained stable. Furthermore, we detected more proliferating and fewer apoptotic Mφ within the interstitium in IFN-γR-deficient MRL-Faslpr mice. Using unilateral ureteral ligation we established that IFN-γR signaling does not alter Mφ recruitment into the kidney. Thus, the increase in Mφ elicited by Mφ growth factors in IFN-γR-deficient MRL-Faslpr mice is a result of enhanced proliferation and decreased apoptosis, and is independent of recruitment. Taken together, we suggest that IFN-γ provides a negative regulatory pathway capable of limiting Mφ-mediated renal inflammation.
A Noncomitogenic CD2R Monoclonal Antibody Induces Apoptosis of Activated T Cells by a CD95/CD95-L-Dependent Pathway
Authors: Fournel Sylvie , Robinet Eric , Bonnefoy-Bérard Nathalie , Assossou Olga , Flacher Monique , Waldmann Herman , Bismuth Georges , Revillard Jean-Pierre
Published: January 2023

Clonal expansion of activated T and B cells is controlled by homeostatic mechanisms resulting in apoptosis of a large proportion of activated cells, mostly through interaction between CD95 (Fas or Apo-1) receptor and its ligand CD95-L. CD2, which is considered as a CD3/TCR alternative pathway of T cell activation, may trigger activation-induced cell death, but the role of CD95/CD95-L interaction in CD2-mediated apoptosis remains controversial. We show here that the CD2R mAb YTH 655.5, which does not induce comitogenic signals when associated with another CD2 mAb, triggers CD95-L expression by preactivated but not resting T cells, resulting in CD95/CD95-L-mediated apoptosis. The critical role of CD95/CD95-L interaction was supported by complete inhibition in the presence of the antagonist CD95 mAb ZB4 and by blocking CD95-L synthesis and surface expression by cycloheximide, cyclosporin A, EGTA, or cytochalasin B. YTH 655.5 was shown to stimulate p56lck phosphorylation and enzymatic activity. However, p56lck activation is not sufficient to trigger apoptosis, because other CD2R and CD4 mAbs that activate p56lck do not induce apoptosis. In conclusion, CD2 can mediate nonmitogenic signals, resulting in CD95-L expression and apoptosis of CD95+ cells.
Repeated Intratracheal Instillations of Nonreplicating Adenovirus 2 Vector Attenuate CTL Responses and IFN-γ Production
Authors: Pascual David W. , Walters Nancy , Hillemeyer Peter
Published: January 2023

The proposed usage of replication-deficient adenovirus (Ad) vectors for corrective gene therapy or for mucosal immunization has been limited in part by the host reactivity to the Ad vector, thus limiting repeated Ad instillations. We have recently shown that the reactivity to the Ad vector is in large part due to increased CD4+ Th1 and Th2 responses as well as elevated IgG and mucosal IgA responses. It has been recently proposed that the diminution of transgene expression in respiratory epithelia was due to increased CTL reactivity to expressed Ad proteins. Herein, we report that repeated intratracheal delivery of a second generation Ad2 vector into mice results in no detectable CTL activity in freshly isolated lymphoid cells from lungs, lower respiratory lymph nodes, or spleens or after in vitro restimulation. In contrast, a single dose of Ad2 vector did elicit a robust CTL response. This attenuation of CTL activity was long lived and was not affected by macrophage depletion or due to a reduction in CD4+ or CD8+ T cells. Examination of cytokine production via MHC class I or class II restimulation by lymphoid cells from three intratracheally treated mice showed an attenuation in the production of IFN-γ by as much as 110-fold. This reduction in IFN-γ could not be attributed to increased IL-4 or IL-10 production. Thus, this study shows that the CTL response to Ad vectors is attenuated upon repeated administration.
Induction of In Vitro Human Macrophage Anti-Mycobacterium tuberculosis Activity: Requirement for IFN-γ and Primed Lymphocytes
Authors: Bonecini-Almeida M. Gloria , Chitale Sadhana , Boutsikakis Iosif , Geng Jiayuan , Doo Howard , He Suhui , Ho John L.
Published: January 2023

Mycobacterium tuberculosis (Mtb) is the world’s leading infectious cause of mortality. Despite the overwhelming data supporting the critical role of cellular immunity, little is known of the early microbial and immune cell interactions and whether human macrophages can be activated to express anti-Mtb activity. We report the reconstitution of an in vitro system whereby human macrophages express anti-Mtb activity only in coculture with PBL and with IFN-γ. Omission of IFN-γ in the cocultures or Mtb lysate/IFN-γ-primed lymphocytes was associated with high growth of Mtb, high IL-10 and IL-12 p40, nearly undetectable IL-12 p70 levels, and the highest percentages of CD4 and CD8 T cells. In contrast, IFN-γ treatment of cocultures containing Mtb lysate/IFN-γ-primed PBL reduced bacilli count by ∼2.5 log, decreased the production of IL-10 by 5.7-fold, increased IL-12 p70 by ∼50-fold, and reduced the percentages of CD4 and CD8 T cells. Activation of anti-Mtb activity was time and dose dependent. At 2000 U/ml of IFN-γ, bactericidal activity was achieved (10-fold reduction from initial inoculum). Anti-Mtb activity against several strains of M. tuberculosis (H37Ra and H37Rv, and C, a clinical isolate) was observed and was associated with expression of inducible nitric oxide synthase. These data suggest that induction of human macrophage anti-Mtb activity required dual signaling from PBL and IFN-γ. Thus, the development of an in vitro human system may greatly facilitate studies to delineate immune cells, cytokines, and effector functions/genes critical in controlling Mtb. Defining the mechanisms may also provide novel treatment strategies for tuberculosis.
Regulation of Human Mast Cell β-Tryptase: Conversion of Inactive Monomer to Active Tetramer at Acid pH
Authors: Ren Shunlin , Sakai Kentaro , Schwartz Lawrence B.
Published: January 2023

At neutral pH, human mast cell β-tryptase is stabilized in its enzymatically active, tetrameric form by heparin, and resists inhibition by biologic protease inhibitors. After dissociation of β-tryptase from heparin, active tetramers rapidly convert to inactive monomers in an isotonic, neutral pH environment. Although reversible transition states probably exist during this conversion, once inactive monomers form, addition of heparin fails to reconstitute active tetramer at neutral pH. The current study shows that complete reactivation of inactive monomers can occur at acidic pH in a heparin-independent manner. The respective rate-determining steps for formation of tetramer and active enzyme from inactive monomers exhibit second and first order kinetics based on an analysis of initial reaction rates. The optimal pH for tetramer formation and reactivation is about 6, suggesting His residues play a critical role. The optimal ionic strength equivalent is 160 mM NaCl; and the optimal temperature range is 22°C to 37°C. We propose a sequential three-step reactivation process at acidic pH, dimerization of monomers (rate-determining second order step), rapid formation of inactive tetramers, and slow formation of active tetramers (overall rate-determining first order step). Whether reactivation of human β-tryptase occurs at extracellular or intracellular sites, where the pH is acidic in vivo, should be considered.
An Immunodominant Epitope from Mycobacterial 65-kDa Heat Shock Protein Protects Against Pristane-Induced Arthritis
Authors: Thompson Stephen J. , Francis James N. , Siew L. Khai , Webb Ginette R. , Jenner Peter J. , Colston M. Joseph , Elson Christopher J.
Published: January 2023

Previous studies showed that mice with pristane-induced arthritis (PIA) and those protected from the disease by preimmunization with mycobacterial 65-kDa heat shock protein (hsp65) possess raised immune responses to hsp65. Additionally, T cells from hsp65-protected mice, but not from pristane-injected or normal mice, produced the Th2-associated cytokines IL-4, IL-5, and IL-10 in response to stimulation with hsp65. Here we demonstrate that the specificity of the immune response to hsp65 and related heat shock protein (hsps) differs between protected and PIA mice. T cells from hsp65-protected mice respond to the bacterial hsps tested but not to the mammalian homologue, hsp58. Similarly, they exhibit high serum titers of anti-hsp65 Abs, yet they have virtually undetectable levels of anti-hsp58 IgG. By contrast, both cellular and humoral immune responses are detectable to bacterial and mammalian hsps in mice with PIA. An immunodominant T cell epitope has been identified in hsp65-protected mice corresponding to amino acids 261–271 from hsp65. Immunization of mice, either before or after the induction of arthritis, with this bacterial peptide, but not its mammalian homologue, protects mice from the development of PIA, and protection is associated with the production of Th2-type cytokines. Other experiments revealed that T cells primed with bacterial 261–271 or the mammalian homologue do not cross-react at the proliferative or cytokine level. These results demonstrate that an hsp65 peptide-specific Th2 response confers protection from PIA but do not support the idea that protection is mediated by a cross-reaction with self hsp58 in the joints.
A Comparison of Signaling Requirements for Apoptosis of Human B Lymphocytes Induced by the B Cell Receptor and CD95/Fas
Authors: Graves Jonathan D. , Draves Kevin E. , Craxton Andrew , Krebs Edwin G. , Clark Edward A.
Published: January 2023

To define how the signaling pathways that mediate the B cell receptor (BCR) death pathway differ from those responsible for CD95/Fas-mediated death, we compared the BCR and Fas death pathways in two human B cell lines, B104 and BJAB. Both BCR- and Fas-induced apoptosis are blocked by the peptide cysteine protease inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD (mlz)), demonstrating a common requirement caspase activity. Despite this common characteristic, the ability of actinomycin D and cycloheximide to block BCR-induced apoptosis, but not apoptosis induced by Fas cross-linking, suggests that a major difference between these two pathways is their differential requirements for new gene and protein synthesis. BCR- and Fas-mediated apoptosis are both accompanied by activation of stress-activated protein kinase and p38 mitogen-activated protein kinase (MAPK). Activation of both stress-activated protein kinase and p38 MAPK was inhibited by ZVAD (mlz), suggesting the involvement of caspases. To determine the role of p38 MAPK activation in BCR- and Fas-induced apoptosis, we employed SB203580, a specific inhibitor of p38 MAPK. SB203580 inhibited BCR-induced apoptosis, but not apoptosis induced by cross-linking Fas. Furthermore, both actinomycin D and SB203580 inhibited BCR-induced, but not Fas-induced, activation of caspase. Collectively, these findings establish a role for p38 MAPK in BCR-induced apoptosis both upstream and downstream of caspase activity. The p38 MAPK pathway may function to regulate transcriptional or translational events that are critical for BCR-induced apoptosis.
Systemic Administration of Endotoxin Induces Bronchopulmonary Hyperreactivity Dissociated from TNF-α Formation and Neutrophil Sequestration into the Murine Lungs
Authors: Lefort Jean , Singer Monique , Leduc Dominique , Renesto Patricia , Nahori Marie Anne , Huerre Michel , Créminon Christophe , Chignard Michel , Vargaftig B. Boris
Published: January 2023

Bronchopulmonary hyperreactivity (BHR), an increased responsiveness to nonspecific bronchoconstrictor agents, is a well-known characteristic of bronchial asthma. It has been recently suggested that the severity of this disease is related to the endotoxin content of house dust. In the present report, it is shown that the i.p. administration of bacterial LPS to mice is followed by a marked early dose-dependent BHR in response to methacholine. The microscopic examination showed no ultrastructural lesions of the lungs or of the airways, but a marked neutrophil accumulation in the capillaries, as confirmed by an increase of the lung content in the neutrophil enzyme marker myeloperoxidase. In parallel, high levels of TNF-α were found in plasma as well as its transcripts in the lung tissues. Using immunologic (anti-TNF-α and anti-granulocyte Abs), and pharmacologic (dexamethasone and vinblastine) tools, it is demonstrated that BHR is apparently neither related to the presence of neutrophils in the pulmonary microvasculature nor to the synthesis of TNF-α.
Allelic Differences in the Relationship Between Proteasome Activity and MHC Class I Peptide Loading
Authors: Benham Adam M. , Grommé Monique , Neefjes Jacques
Published: January 2023

MHC class I molecules are cell surface glycoproteins that play a pivotal role in the response to intracellular pathogens. The loading of MHC class I molecules with antigenic substrates takes place in the endoplasmic reticulum. This requires a functional TAP transporter, which translocates peptides into the endoplasmic reticulum from the cytosol. The generation of antigenic peptides from polypeptide precursors is thought to be mediated in the cytosol by the proteasome. Previously, we have demonstrated that inhibiting the proteasome with the specific covalent inhibitor lactacystin results in a direct reduction of peptide-loaded MHC class I molecules. This indicates that the proteasome is the limiting step in the MHC class I pathway. In this study we use isoelectric focusing to demonstrate that two related MHC class I alleles, HLA-A3 and HLA-A11, as well as HLA-B35 do not follow this behavior. In contrast to other class I alleles expressed by the same cells, these alleles are loaded with peptides and mature normally when proteasome activity is severely inhibited. Our observations highlight a new level of diversity in the MHC class I system and indicate that there are allele-specific differences in the linkage between proteasome activity and MHC class I peptide loading.
Cutting Edge: BASH, A Novel Signaling Molecule Preferentially Expressed in B Cells of the Bursa of Fabricius
Authors: Goitsuka Ryo , Fujimura Yu-ichi , Mamada Hiroshi , Umeda Akiko , Morimura Toshifumi , Uetsuka Koji , Doi Kunio , Tsuji Sachiyo , Kitamura Daisuke
Published: January 2023

The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.
Cutting Edge: Identification of the 2B4 Molecule as a Counter-Receptor for CD48
Authors: Latchman Yvette , McKay Paul F. , Reiser Hans
Published: January 2023

The CD48 molecule belongs to a subfamily of the Ig superfamily that also includes the CD2, CD58, 2B4, Signaling lymphocyte activation molecule (SLAM), and Ly-9 molecules. Receptor-ligand interactions are known to occur between several members of this family, and these interactions can strengthen cell to cell adhesion. In mice, the CD48 molecule can bind to CD2. To search for additional ligands of murine CD48, we have generated a chimeric fusion protein consisting of the extracellular domain of murine CD48 and the C region of human IgG1. The results of immunofluorescence and immunoprecipitation experiments in which this reagent was used identify the 2B4 molecule as a novel counter-receptor of CD48.
Cutting Edge: Differentiation of Human NK Cells into NK1 and NK2 Subsets
Authors: Peritt David , Robertson Susan , Gri Giorgia , Showe Louise , Aste-Amezaga Miguel , Trinchieri Giorgio
Published: January 2023

Human NK cells cultured in the presence of IL-12 or IL-4 differentiate into cell populations with distinct patterns of cytokine secretion similar to Th1 and Th2 cells. NK cells grown in IL-12 (NK1) produce IL-10 and IFN-γ, whereas NK cells grown in IL-4 (NK2) produce IL-5 and IL-13. Although these NK cell subsets do not differ in cytotoxic activity, NK1 cells express higher levels of cell surface CD95 (Fas) Ag than NK2 cells and are more sensitive to Ab or chemically induced apoptosis. Like Th1 cells, NK1 cells accumulate much higher levels of the IL-12Rβ2-chain mRNA and are significantly more responsive to IL-12 than NK2 cells at the level of activation of STAT4 transcription factor. The identification of NK cell subsets that are analogous to T cell subsets suggests a new role for NK cells in innate inflammatory responses and in their effect on adaptive immunity.
B Cell Responses to a Peptide Epitope. VII. Antigen-Dependent Modulation of the Germinal Center Reaction
Authors: Agarwal Anshu , Nayak Bishnu P. , Rao Kanury V. S.
Published: January 2023

Germinal center responses to two analogous peptides, PS1CT3 and G32CT3, that differ in sequence only at one position within the B cell epitopic region were examined. In comparison with peptide PS1CT3, peptide G32CT3 elicited a poor germinal center response. By demonstrating equal facility of immune complexes with IgM and IgG Ab isotypes to seed germinal centers, we excluded differences in isotype profiles of early primary anti-PS1CT3 and anti-G32CT3 Ig as the probable cause. Quantitative differences in germinal center responses to the two peptides were also not due to either qualitative/quantitative differences in T cell priming or variation in the frequency of the early Ag-activated B cells induced. Rather, they resulted from qualitative differences in the nature of B cells primed. Analysis of early primary anti-PS1CT3 and anti-G32CT3 IgMs revealed that the latter population was of a distinctly lower affinity, implying the existence of an Ag affinity threshold that restricts germinal center recruitment of G32CT3-specific B cells. The impediment in anti-G32CT3 germinal center initiation could be overcome by making available an excess of Ag-activated Th cells at the time of immunization. This resulted in the appearance of a higher affinity population of G32CT3-specific B cells that, presumably, are now capable of seeding germinal centers. These data suggest that the strength of a germinal center reaction generated is Ag dependent. At least one regulatory parameter represents the quality of B cells that are initially primed.
In Vivo Anergized CD4+ T Cells Have Defective Expression and Function of the Activating Protein-1 Transcription Factor
Authors: Sundstedt Anette , Dohlsten Mikael
Published: January 2023

The transcription factor activating protein-1 (AP-1) contributes significantly to the regulation of IL-2 gene expression during T cell activation and has been suggested to play a unique role in T cell anergy in vitro. In this study we have used the superantigen staphylococcal enterotoxin A to investigate the regulation of AP-1 in T cell anergy in vivo. Repeated injections of staphylococcal enterotoxin A induce a state of anergy in CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. The perturbed IL-2 response in anergic T cells correlated with reduced DNA binding activity of the transcription factors AP-1 and Fos/Jun-containing NF-AT. Using AP-1-luciferase reporter transgenic mice, we now demonstrate the lack of AP-1-dependent transcription. AP-1 activity is controlled by synthesis of its subunits Fos and Jun and by posttranslational phosphorylations. Analysis of Fos and Jun protein levels revealed no major differences in the expression of Jun proteins, but a marked decrease in c-Fos in anergic T cells. Experiments in transgenic mice overexpressing c-Fos (H2-c-fos) showed reconstituted AP-1 DNA binding. In contrast, the AP-1-driven transcription and IL-2 production remained suppressed. The Jun N-terminal kinase is known to play a critical role in regulating AP-1 trans-activation. Analyses of Jun N-terminal kinase demonstrated normal protein amounts, but reduced enzymatic activity, in anergic compared with activated CD4+ T cells. This suggests that in vivo anergized T cells have defects in the AP-1 pathway due to both reduced protein expression and perturbed posttranslational modifications.
Somatic Mutation in the Neonatal Mouse
Authors: Giorgetti Carol A. , Press Joan L.
Published: January 2023

Several mechanisms that diversify the adult immune repertoire, such as terminal deoxynucleotidyl transferase-dependent N region addition, are not available to the neonatal mouse. One important process that contributes to protective immunity in the adult is somatic mutation, which plays a major role in the generation of high affinity memory B cells. It is not clear whether B cells in the neonatal mouse can activate the somatic mutation machinery. To investigate this, we immunized neonates with poly(l-Tyr,l-Glu)-poly-d, l-Ala–poly-l-Lys complexed with methylated BSA, or (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken γ-globulin. Eight to fourteen days after priming, V(D)J rearrangements of known VH genes (VHSM7 family) were screened for mutations using a temperature-melt hybridization assay and oligonucleotide probes specific for complementarity-determining regions I and II; possible mutations were confirmed by sequence analysis. More mutations per sequence were found in heavy chains from neonates immunized with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken γ-globulin than in those from neonates immunized with poly(l-Tyr, l-Glu)-poly-d,l-Ala-poly-l-Lys complexed with methylated BSA. Mutations were found in heavy chains lacking N regions, suggesting that B cells of the putative fetal lineage can somatically mutate and diversify an initially limited repertoire. Since neonates immunized as early as 1 or 2 days after birth had mutations, the somatic mutation machinery can be activated soon after birth, suggesting that early vaccination should result in affinity maturation and protective immunity in the neonate.
OPG/FDCR-1, a TNF Receptor Family Member, Is Expressed in Lymphoid Cells and Is Up-Regulated by Ligating CD40
Authors: Yun Theodore J. , Chaudhary Preet M. , Shu Geraldine L. , Frazer J. Kimble , Ewings Maria K. , Schwartz Stephen M. , Pascual Virginia , Hood Leroy E. , Clark3 Edward A.
Published: January 2023

We have cloned a TNFR family member from a follicular dendritic cell (FDC)-like cell line, FDC-1. This molecule, FDC-derived receptor-1 (FDCR-1), is identical to osteoprotegerin (OPG), a soluble cytokine that regulates osteoclast differentiation. Recently, OPG/FDCR-1 has been characterized as a second receptor for receptor activator of NF-κB ligand (RANKL)/TNF-related activation-induced cytokine (TRANCE), a primarily T-cell restricted TNF family member that augments dendritic cell (DC) function. In this report, we demonstrate that OPG/FDCR-1 is membrane bound on the surface of transfected baby hamster kidney (BHK) and untransfected FDC-1 cells. We also found a restricted OPG/FDCR-1 expression pattern in lymphoid cells, specifically in B cells, DCs and FDC-enriched fractions, which in B cells and DCs is up-regulated by CD40 stimulation. Because OPG/FDCR-1 shares some properties with RANK, the first RANKL/TRANCE receptor, we discuss how the balance between RANK and OPG/FDCR-1 expression could influence immune responses and, ultimately, germinal center formation.
Isolated Human Germinal Center Centroblasts Have an Intact Mismatch Repair System
Authors: Park Kyoungsook , Kim Jaehoon , Kim Han-Soo , Shin Hyun S.
Published: January 2023

Ig somatic hypermutation contributes to the generation of high-affinity Abs that are essential for efficient humoral defense. The presence of multiple point mutations in rearranged Ig V genes and their immediate flanking sequences suggests that the DNA repair system may not be working properly in correcting point mutations introduced to the restricted region of Ig genes. We examined the DNA repair functions of germinal center (GC) centroblasts, which are the cells in which ongoing Ig hypermutation takes place. We found that GC centroblasts express all known components of the human DNA mismatch repair system, and that the system corrects DNA mismatches in a strand-specific manner in vitro. We conclude that general suppression of mismatch repair at the cellular level does not occur during somatic hypermutation.
The Importance of TGF-β in Murine Visceral Leishmaniasis
Authors: Wilson Mary E. , Young Betty M. , Davidson Beverly L. , Mente Kimberly A. , McGowan Stephen E.
Published: January 2023

IFN-γ is critical for the cure of leishmaniasis in humans and mice. BALB/c mice are genetically susceptible to infection with the visceralizing species of Leishmania, L. chagasi. We have evidence that a soluble factor(s) inhibits IFN-γ production by cultured liver granuloma cells from BALB/c mice during L. chagasi infection. In contrast, liver granulomas from C3H.HeJ mice, which are genetically resistant to L. chagasi infection, produce abundant IFN-γ. According to ELISAs and neutralization studies, there was not evidence that the Th2-type cytokines IL-10 or IL-4 contributed to IFN-γ suppression. However, both Ab neutralization and immunohistochemistry showed that granuloma-derived TGF-β was, at least in part, responsible for inhibiting IFN-γ release by CD4+ cells in BALB/c liver granuloma cultures. Consistently, TGF-β levels were high in liver granulomas from susceptible BALB/c mice but low in resistant C3H mice or in BALB/c mice that were immunized against L. chagasi disease. Administration of recombinant adenovirus expressing TGF-β (AdV-TGFβ) but not IL-10 (AdV-IL10) caused genetically resistant C3H mice to become significantly more susceptible to L. chagasi infection. In contrast, either AdV-TGFβ or AdV-IL10 could abrogate the protective immune response achieved by immunization of BALB/c mice. We conclude that locally secreted TGF-β inhibits Th1-associated cure of murine visceral leishmaniasis caused by L. chagasi, independently of Th2-type cytokines.
Production of Proinflammatory Cytokines by Phorbol Myristate Acetate-Treated THP-1 Cells and Monocyte-Derived Macrophages After Phagocytosis of Apoptotic CTLL-2 Cells
Authors: Kurosaka Kahori , Watanabe Naoko , Kobayashi Yoshiro
Published: January 2023

Because it is generally believed that apoptosis is not associated with inflammation, we hypothesized that the interaction of phagocytes with apoptotic cells provides a negative or null signal for inflammation. However, we recently found that the interaction led to the production of proinflammatory cytokines but not antiinflammatory cytokines, although the apoptotic cell membranes appeared to be intact. In this study, we examined in detail the relationship among the kinetics of apoptosis, phagocytosis and production of cytokines by macrophages. Among the time points examined, murine CTLL-2 cells became apoptotic in terms of cell size and exposure of phosphatidylserine after 12 h of culture in the absence of IL-2, and at the same time they began to be phagocytosed and lead to proinflammatory cytokine production by PMA-treated THP-1 cells (human macrophages). The phagocytosis of apoptotic cells by macrophages was also confirmed by confocal laser microscopy. The coculturing of human macrophages with murine apoptotic cells led to the production of human proinflammatory cytokines, notably IL-8, at both the mRNA level and the protein level. The coculturing of monocyte-derived macrophages with the apoptotic cells also led to the production of IL-8 protein. Both the phagocytosis and production of the cytokines were suppressed by either phospho-l-serine or RGDS (Arg-Gly-Asp-Ser), but not by RGES (Arg-Gly-Glu-Ser). Thus, the production of proinflammatory cytokines and phagocytosis of apoptotic CTLL-2 cells appear to be closely interrelated.