bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of C3b/C3d binding sites and factor I cofactor regions within mouse complement receptors 1 and 2.
Authors: Molina H , Kinoshita T , Webster C B , Holers V M
Published: January 2023

Human and murine CR1 and CR2 are defined as evolutionary homologues on the basis of their in vitro activities and a shared structural motif known as a short consensus repeat (SCR). To identify additional similarities between the two species, we analyzed the functional domains within the mouse receptors by constructing mouse-human chimeric cDNAs in which the C3 binding site of human CR2 has been replaced by different regions within the first eight SCRs of mouse CR1. Rosette analysis of cells expressing chimeric proteins, with erythrocytes bearing different mouse C3 fragments, coupled with rosette inhibition studies using specific anti-mouse CR1/CR2 mAbs reveal a weak C3b binding site within SCRs 1 and 2 of mouse CR1. There is no independent C3b interacting domain within SCRs 3 to 6, but their presence enhances C3 binding. A molecule that contains only the first six SCRs of mouse CR1 also binds C3b, but with less efficiency. There is no C3d binding area within the first six SCRs, but our data confirms previous studies indicating an additional C3b/C3d binding region within SCRs 7 and 8 of mouse CR1 (SCRs 1-2 of mouse CR2). The presence of SCRs 1 to 4 is required for C3 cofactor activity. 8C12, a mAb which blocks C3b erythrocyte rosette binding and the C3 cofactor activity of mouse CR1, binds only to chimeras containing SCRs 3 to 4.(ABSTRACT TRUNCATED AT 250 WORDS)
Transforming growth factor beta 1 costimulated growth and regulatory function of staphylococcal enterotoxin B-responsive CD8+ T cells.
Authors: Rich S , Seelig M , Lee H M , Lin J
Published: January 2023

Transforming growth factor beta (TGF-beta) exhibits diverse effects on growth and differentiation of a wide range of cell types. In the immune system, TGF-beta 1 is a potent inhibitor of T cell proliferation and certain T cell effector functions. However, TGF-beta 1 also enhances growth of T cells, predominantly of naive phenotype, and induces their expression of selected cytokines. We have previously demonstrated that TGF-beta 1 costimulates growth of highly purified murine CD8+ T cells activated by immobilized anti-CD3 Ab. TGF-beta 1-costimulated CD8+ T cells rapidly express a memory phenotype, lose lytic function, and express a mixed cytokine pattern with IL-2, IFN-gamma, and appreciable IL-10, as well as TGF-beta 1. The present work examines the possibility that TGF-beta 1 similarly costimulates response of murine CD8+ T cells to the microbial superantigen staphylococcal enterotoxin B (SEB) and characterizes their effector and regulatory functions. TGF-beta 1 significantly enhances CD8+ T cell proliferation to SEB in the presence of MHC class II-positive APC and TGF-beta 1-primed CD8+ T cells are enriched for SEB-reactive V beta 8+ TCR expression. TGF-beta 1 priming also up-regulates a memory-like CD45RBlowCD44highMEL-14low phenotype. TGF-beta 1 priming inhibits development of SEB-specific lytic effector function by more than 90%. However, TGF-beta 1-primed CD8+ effector T cells express elevated levels of IL-10 and TGF-beta 1, variable IFN-gamma, and undetectable IL-4. Additionally, they exhibit growth inhibitory effector function of SEB-induced proliferation of other CD4+ and CD8+ T cells. Growth inhibition by TGF-beta 1-primed CD8+ T cells is reversed in part by anti-IL-10 Ab. Thus, in the context of SEB response, TGF-beta 1 promotes the outgrowth and induces the effector function of CD8+ T cells that have the capacity to impair T cell clonal growth.
Activation of lymphocyte function-associated molecule-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) mimicked by an antibody directed against CD18.
Authors: Petruzzelli L , Maduzia L , Springer T A
Published: January 2023

The beta 2-integrin (CD18) family members bind to their ligands subsequent to activation of a number of well defined and diverse signal transduction pathways. The precise molecular changes associated with activation of the integrin family members have remained elusive. Here, we characterize a monoclonal, CBR LFA-1/2, that binds to the beta 2-subunit and is able to mimic activation induced upon stimulation by phorbol esters. The Ab induces binding of the LFA-1-expressing cell line, JY, to ICAM-1 (CD54) and ICAM-3 (CD50). Activation of binding by this Ab is independent of Fc interactions and does not occur through cross-linking at the cell surface, because the Fab fragment of the Ab is able to modulate the same effect. Stimulation of neutrophils with CBR LFA-1/2 induces binding to ICAM-1 through activation of both LFA-1 and Mac-1. Activation of Mac-1 by CBR LFA-1/2 was further confirmed by stimulation of neutrophil binding to fibrinogen, a ligand for Mac-1. CBR LFA-1/2 lowers by 10-fold the concentration of Mg2+ required to achieve maximal binding of LFA-1 to ICAM-1. It therefore appears that CBR LFA-1/2 induces a conformational change that directly increases the avidity of beta 2-integrins for ligands.
Induction of IL-10 synthesis by human keratinocytes through CD23 ligation: a cyclic adenosine 3',5'-monophosphate-dependent mechanism.
Authors: Bécherel P A , LeGoff L , Francès C , Chosidow O , Guillosson J J , Debré P , Mossalayi M D , Arock M
Published: January 2023

Ligation of the low affinity receptor for IgE, CD23/Fc epsilonRII, in human keratinocytes (HK) and monocytes induces the synthesis of proinflammatory cytokines (IL-6 and TNF-alpha), partly under the dependence of cAMP and nitric oxide pathways. Moreover, CD23 ligation induces IL-10 production in human monocytes. Since synthesis of IL-10 by HK is still a matter of debate, we investigate whether keratinocytes could produce IL-10 upon CD23 stimulation. Here, our data show that CD23 ligation induces significant IL-10 synthesis in HK, a phenomenon inhibited by cAMP antagonists, but not by inhibitors of the nitric oxide pathway. Accordingly, cAMP agonist induced significant IL-10 synthesis by HK, while nitric oxide-releasing chemical did not. Treatment of HK with anti-IL-10 mAb potentiated their CD23-mediated TNF-alpha synthesis. These data indicate that engagement of surface CD23 on human keratinocytes induces the synthesis of IL-10, which, in turn, down-regulates their proinflammatory response.
Complex origin of the HLA-DR10 haplotype.
Authors: Gongora R , Figueroa F , Klein J
Published: January 2023

The region of the HLA complex occupied by the DRB genes has undergone many rearrangements in the course of primate evolution. The rearrangements have produced a number of haplotypes differing from one another in the number and composition of the DRB genes. Some of the rearrangements also affected the DRB genes themselves. Selective intron sequencing has revealed the DR10 haplotype to be composed of at least three segments, each of different origin. The haplotype carries three DRB genes (gene fragments): DRB1*10, DRB6, and DRB9. The 5' end of the DRB1*10 gene, from the promoter region to a site in intron 1 approximately 500 bp from the beginning of exon 2, is derived from a DRB1*03-like gene. The segment of the DR10 haplotype encompassing the rest of the DRB1*10 gene and extending to the region between the DRB1 and DRB6 genes is of independent origin; it diverged from other DRB genes (DRB1*01 and DRB1*03) approximately 30 million years ago. Finally, the third segment encompassing the remainder of the DR10 haplotype is derived from a DR1-like haplotype. Since the functional part of the DR10 haplotype is of independent origin, there is little justification for the currently common practice of placing the haplotype together with DR1 in the group of DR1 haplotypes. The rearrangements in the DR haplotypes may constitute one of several mechanisms for increasing diversity at the DRB loci. The region of high instability seems to be flanked by conservatively evolving regions.
Second-hand smoke is an adjuvant for T helper-2 responses in a murine model of allergy.
Authors: Seymour B W , Pinkerton K E , Friebertshauser K E , Coffman R L , Gershwin L J
Published: January 2023

Epidemiologic studies have suggested that "second-hand smoke" increases the incidence and severity of allergies and asthma. To address this, we developed a murine model that included generation of and exposure to environmental tobacco smoke (ETS) followed by aerosolized Ag challenge. We report herein that exposure of OVA-sensitized BALB/c mice to ETS elicits a rapid and prolonged exaggerated response with respect to IgE, IgG1, eosinophils, and Th2 cytokines (particularly IL-4 and IL-10). There was a significant increase (p = 0.0159) in the total cells of the bronchoalveolar lavage in OVA-sensitized mice exposed to ETS when compared with those in ambient air. This is the first experimental data to demonstrate that "second-hand smoke" up-regulates the allergic response to inhaled Ag. The results have important health implications for children and allergic individuals residing in households with smokers.
Diminished class II-associated Ii peptide binding to the juvenile dermatomyositis HLA-DQ alpha 1*0501/DQ beta 1*0301 molecule.
Authors: Reed A M , Collins E J , Shock L P , Klapper D G , Frelinger J A
Published: January 2023

HLA class II molecules bind and present peptide Ags to T cells, binding specific sets of peptides due to polymorphism in the peptide binding groove. Class II proteins associate with the invariant chain (Ii chain) and its derived class II-associated Ii peptide (CLIP). Ii chain association is important for normal trafficking of class II proteins to the peptide loading vesicles and for blocking premature access of peptides to HLA class II molecules during maturation. We have previously shown that juvenile dermatomyositis is associated with the HLA-DQA1*0501 allele. There is limited information available about the interaction of any DQ molecule with the Ii chain and little information about binding of individual peptides to HLA-DQalpha1*0501/DQbeta1*0301. We sequenced peptides eluted from the juvenile dermatomyositis-associated class II allele HLA-DQalpha1*0501/DQbeta1*0301. Surprisingly, we found no Ii chain or CLIP. Further examination of peptide binding to the HLA-DQalpha1*0501/DQbeta1*0301 molecule demonstrated poor CLIP binding. However, newly synthesized HLA-DQalpha1*0501/DQbeta1*0301 molecules do associate with intact Ii chain. Molecular modeling suggests that CLIP binds differently to HLA-DQalpha1*0501/DQbeta1*0301 than to DR molecules. The lack of CLIP association suggests that HLA-DQalpha1*0501/DQbeta1*0301 has access to peptides earlier in the processing pathway and so might encounter novel peptides that induce autoimmunity.
Ion channels, Ca2+ signaling, and reporter gene expression in antigen-specific mouse T cells.
Authors: Kerschbaum H H , Negulescu P A , Cahalan M D
Published: January 2023

Whole cell recordings were performed in parallel with measurements of intracellular Ca2+ ([Ca2+]i) and gene expression using the murine T cell hybridoma, B3Z, a cell line stably infected with a lacZ reporter gene, driven by the minimal IL-2 promoter (NF-AT, nuclear factor of activated T cells). The physiologic roles of ion channels in B3Z cells were investigated by correlating the pharmacology of channel block with [Ca2+]i, and expression of lacZ. In B3Z cells and activated human T cells, the major component of voltage-gated K+ (K(V)) current had biophysical and pharmacologic properties associated with type n channels encoded by Kv1.3; a minor K(V) component was charybdotoxin (CTX) resistant. Ca2+-activated K+ (K(Ca)) current was sensitive to CTX, but not to margatoxin (MgTX). Inwardly rectifying K+ (K(IR)) current was blocked completely by 200 microM of Ba2+. Outwardly rectifying Cl- currents were induced by cell swelling. An inwardly rectifying Ca2+ current (I(CRAC)) was activated by dialyzing the cell with 10 mM EGTA and 10 microM IP3. CTX reduced thapsigargin-stimulated [Ca2+]i signaling and gene expression by approximately 25%. Although the thapsigargin-stimulated [Ca2+]i signal was resistant to complete inhibition by K+ channel blockers, it was very sensitive to the K+ diffusion potential and Cl- removal, suggesting that drug-resistant K+ channels and perhaps Cl- channels can maintain a sufficiently negative membrane potential to drive Ca2+ influx. Neither [Ca2+]i signaling nor gene expression induced by stimulation of the CD3-epsilon subunit of the TCR was inhibited by ion channel blockers used in this study. We conclude that several channel types can contribute to maintenance of Vm, Ca2+ signals, and gene expression.
Mechanisms of L-selectin regulation by activated T cells.
Authors: Chao C C , Jensen R , Dailey M O
Published: January 2023

The activation of T cells through the TCR results in the differential regulation of a set of adhesion molecules that dramatically alters lymphocyte migration and tissue localization properties in vivo. L-selectin, the lymph node homing receptor, is central to the control of lymphocyte recirculation. We examined the regulation of L-selectin as a function of time after activation in vitro. Within an hour of stimulation, T cells down regulate L-selectin, with a 90% loss by 4 h, due to accelerated proteolytic cleavage. Over the course of the following 48 h, surface receptor expression increases markedly. This is due to an increase in L-selectin mRNA, which, in turn, results from increased message stability. During the next several days after activation, L-selectin levels decrease, resulting in L-selectin-negative T cells by 5 to 7 days after stimulation. This decrease occurs faster in CD8 than in CD4 T cells. During this phase of regulation, L-selectin message remains stable even as the level of specific mRNA continuously decreases. This indicates that the L-selectin-negative phenotype of T cells late after activation is due to the down-regulation of gene transcription. These results demonstrate that after stimulation through the TCR, the expression of L-selectin changes in a triphasic pattern, with an initial marked decrease, followed by a transient phase of superinduction and then a loss of expression. These changes are regulated through the complex interactions between several mechanisms at the transcriptional, post-transcriptional, and protein turnover levels.
Recombinant soluble alphabeta T cell receptors protect T cells from immune suppression: requirement for aggregated multimeric, disulfide-linked alphabeta heterodimers.
Authors: Paliwal V , Ptak W , Sperl J , Braswell E , Askenase P W
Published: January 2023

Recombinant soluble T cell receptors (sTCR) protected contact sensitivity (CS) effector T cells from down-regulation or immunosuppression. CS-protecting sTCR were released enzymatically from the surface of thymoma cells transfected with cDNAs encoding TCR-alpha and -beta extracellular domains that were expressed with a phosphatidylinositol linkage. sTCR affinity purified on anti-TCR-alpha and anti-TCR-beta mAb columns had identical CS-protective activity, as did sTCR from a CD4+ Th2 clone or from a CD8+ cytotoxic clone. Reduced sTCR alpha- and beta-chains had no CS-protective activity, but this was restored when the TCR chains were rejoined into disulfide-linked alphabeta heterodimers. sTCR CS protection was Ag nonspecific, MHC unrestricted, and not influenced by the relevant synthetic peptide specific for the TCR complexed with appropriate MHC. CS protection may have resided in the sTCR constant region. When heated at 62 degrees C for 30 min, sTCR formed a CS-protecting aggregate, with a molecular mass of 481 +/- 37 kDa, corresponding to an alphabeta TCR pentamer. HPLC gel filtration essentially confirmed the molecular mass at 516 kDa for the multimer, while the monomer, which was an alphabeta TCR heterodimer, had an expected molecular mass of approximately 104 kDa and no bioactivity. In summary, the pentameric sTCR may bind to and activate lymphoid cells, perhaps via constant domains, resulting in protection of CS effector T cells from down-regulation. The ability of sTCR to protect CS effector T cells from down-regulation/suppression, if generalized, could overcome immunosuppression accompanying infectious diseases, particularly AIDS, or in tumors.
Human endothelial cell activation and mediator release in response to the bacterial exotoxins Escherichia coli hemolysin and staphylococcal alpha-toxin.
Authors: Grimminger F , Rose F , Sibelius U , Meinhardt M , Pötzsch B , Spriestersbach R , Bhakdi S , Suttorp N , Seeger W
Published: January 2023

Escherichia coli hemolysin (HlyA) and Staphylococcus aureus alpha-toxin are membrane-perturbating bacterial exotoxins that have been implicated as significant virulence factors in human diseases. We investigated the capacity of these toxins to cause cell activation and mediator release in human endothelial cells, compared with the efficacies of thrombin and the Ca2+ ionophore A23187. Concentration ranges tested were 1 to 1000 ng/ml (HlyA), 0.01 to 10 micro/ml (alpha-toxin), 0.01 to 10 U/ml (thrombin), and 0.01 to 10 microM (A23187). All stimuli caused dose-dependent generation of platelet-activating factor, nitric oxide, and prostaglandin I2. HlyA and thrombin effected time- and dose-dependent accumulation of large quantities of inositol phosphates, with maximum effects at 100 ng/ml and 1 U/ml, respectively. Corresponding time course and dose dependency were noted for HlyA-elicited diacylglycerol formation. In contrast, only the highest concentrations of alpha-toxin (10 microg/ml) and A23187 (10 microM) effected some moderate inositol phosphate accumulation, and this was suppressed in the presence of the platelet-activating factor antagonist WEB 2086. Metabolic and secretory responses elicited by alpha-toxin were dependent on the presence of extracellular Ca2+. We conclude that both HlyA and alpha-toxin are potent inductors of inflammatory and vasodilatory mediators in human endothelial cells. HlyA-elicited effects may proceed predominantly via activation of the phosphatidylinositol hydrolysis-related signal transduction pathway, whereas transmembrane Ca2+ flux appears to be the major event underlying the release of mediators in response to alpha-toxin. These toxin properties may contribute to vasoregulatory and inflammatory disturbances encountered in states of severe infection and sepsis.
Thrombopoietin promotes adhesion of primitive human hemopoietic cells to fibronectin and vascular cell adhesion molecule-1: role of activation of very late antigen (VLA)-4 and VLA-5.
Authors: Cui L , Ramsfjell V , Borge O J , Veiby O P , Lok S , Jacobsen S E
Published: January 2023

Thrombopoietin (Tpo), the ligand for c-mpl and a principal regulator of megakaryocytopoiesis and platelet production, has been demonstrated to stimulate the growth and differentiation of megakaryocyte as well as multipotent hemopoietic progenitor cells. In the present study we demonstrate that Tpo can stimulate the adhesion of the Mo7e progenitor cell line to fibronectin (Fn) as well as vascular cell adhesion molecule-1 through activation of very late antigen (VLA)-4 and VLA-5, adhesion molecules previously demonstrated to be involved in regulation of steady state hemopoiesis. Tpo-induced adhesion was concentration dependent, reached a maximum following 30 min, and appeared to be dependent on adenylate cyclase, and tyrosine kinase activity. Furthermore, second messenger inhibitors implicated essential and complimentary roles of phosphatidylinositol-3-kinase and protein kinase C in mediating Tpo-induced adhesion. The ability of Tpo to promote adhesion to fibronectin was comparable to that of IL-3, but less than that of stem cell factor. Unlike the ability of these cytokines to synergistically enhance growth of Mo7e as well as normal progenitor cells, no synergy was observed with regard to their ability to enhance adhesion. Finally, Tpo stimulated adhesion of primitive (CD34+ CD38-) human bone marrow cells to fibronectin, predominantly through activation of VLA-5, whereas no such effect could be observed on CD34+ CD38+ bone marrow cells. Thus, Tpo might play an important role in early hemopoiesis, at least in part through its ability to promote adhesion through activation of adhesion molecules on hemopoietic progenitor cells.
Enhanced B-1 Cell Development, But Impaired IgG Antibody Responses in Mice Deficient in Secreted IgM
Authors: Boes Marianne , Esau Christine , Fischer Michael B. , Schmidt Tara , Carroll Michael , Chen Jianzhu
Published: January 2023

The role of endogenous natural IgM in promoting the adaptive Ab response was investigated in newly constructed mutant mice in which B cells do not secrete IgM but still express surface IgM and IgD and undergo class switching to express other Ig isotypes. While the mutant mice had relatively normal numbers of conventional B (B-2) cells in all tissues examined, unexpectedly, B-1 cells in the peritoneum and spleen were approximately threefold more abundant. The elevated levels of B-1 cells were already detectable at 4 wk of age and were stably maintained throughout life. The levels of serum IgG2a, IgG3, and IgA were also elevated in the mutant mice at an early age. IgG2a response to a T cell-independent Ag was augmented, whereas IgG Ab responses to suboptimal doses of a T cell-dependent Ag were impaired. The latter defect was associated with fewer splenic germinal centers, impaired Ab affinity maturation, and less Ag trapping on follicular dendritic cells. Together, these findings demonstrate a physiologic role of natural IgM in the feedback regulation of B-1 cell development, the regulation of IgG2a production, and the promotion of efficient B-2 cell Ab responses.
Dissociation of Proteasomal Degradation of Biosynthesized Viral Proteins from Generation of MHC Class I-Associated Antigenic Peptides
Authors: Antón Luis C. , Snyder Heidi L. , Bennink Jack R. , Vinitsky Alexander , Orlowski Marian , Porgador Angel , Yewdell Jonathan W.
Published: January 2023

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP50–57) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP147–155) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP147–155 complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.
Staphylococcus aureus-Induced Septic Arthritis and Septic Death Is Decreased in IL-4-Deficient Mice: Role of IL-4 as Promoter for Bacterial Growth
Authors: Hultgren Olof , Kopf Manfred , Tarkowski Andrzej
Published: January 2023

Lack of IL-4 has been shown to be protective in some experimental models of infectious diseases in mice such as cutaneous leishmaniasis. At the same time IL-4, together with other Th2 cytokines, including IL-10 and IL-13, is known as an anti-inflammatory cytokine with the potential to down-regulate proinflammatory cytokine production. To investigate the role of IL-4 in experimental Staphylococcus aureus-induced and T lymphocyte-mediated arthritis, IL-4-deficient C57BL/6 mice (IL-4−/−) and their congenic controls (IL-4+/+) were inoculated with a toxic shock syndrome toxin-1-producing S. aureus strain. In IL-4+/+ mice, arthritis peaked 14 days after bacterial inoculation, whereas, at that time, IL-4−/− mice displayed significantly less frequent (p < 0.05) joint inflammation. Paralleling lower frequency of arthritis, IL-4-deficient mice showed a decreased bacterial burden in joints (p = 0.014) and kidneys (p = 0.029), as well as lower infection-triggered weight decrease and mortality. In vitro, IL-4 inhibited intracellular killing of S. aureus in infected macrophages, without affecting phagocytosis. This finding may explain the enhanced staphylococcal clearance observed in IL-4−/− mice in vivo. Our results suggest that IL-4 and IL-4-dependent Th2 responses promote septic arthritis and sepsis-related mortality by inhibition of bacterial clearance during S. aureus infection.
Quercetin Sensitizes RBL-2H3 Cells to Polybasic Mast Cell Secretagogues Through Increased Expression of Gi GTP-Binding Proteins Linked to a Phospholipase C Signaling Pathway
Authors: Senyshyn Jan , Baumgartner Rudolf A. , Beaven Michael A.
Published: January 2023

Polybasic secretagogues such as mastoparan, compound 48/80, substance P, and somatostatin stimulate secretion in rat peritoneal mast cells through direct activation of the heterotrimeric G protein, Gi-3. Cultured RBL-2H3 mast cells do not normally respond to these secretagogues, but, as reported here, they do so after prolonged exposure to the kinase inhibitor, quercetin. This inhibitor, which causes phenotypic changes in RBL-2H3 cells, induces a substantial increase (more than sevenfold) in the expression of α subunits of the pertussis toxin-sensitive G proteins, Gi-2 and Gi-3. Compound 48/80-induced secretion is associated with transient hydrolysis of phosphoinositides and a transient increase in cytosolic calcium ions. These responses are inhibited by pertussis toxin, and in addition, secretion is blocked by calcium chelation and the protein kinase C inhibitor, Ro31-7549. These results delineate a pathway for compound 48/80-induced secretion in mast cells via Gi protein(s), phospholipase C, calcium, and protein kinase C. The results also imply that phospholipase C, most likely phospholipase Cβ3, can be transiently activated in RBL-2H3 cells by subunits of Gi proteins to induce cellular responses.
Killer Cell Inhibitory Receptors for MHC Class I Molecules Regulate Lysis of Melanoma Cells Mediated by NK cells, γδ T Cells, and Antigen-Specific CTL
Authors: Bakker Alexander B. H. , Phillips Joseph H. , Figdor Carl G. , Lanier Lewis L.
Published: January 2023

NK cells and T cells express killer cell inhibitory receptors (KIR) recognizing polymorphic MHC class I molecules. Although prior studies have established that MHC class I can protect normal and transformed hematopoietic cells from NK cell lysis, the role of MHC class I on the recognition of solid tumors has been controversial. In this study, we investigated whether interactions of KIR with their ligands on melanoma tumor cells could inhibit tumor cell lysis by NK and γδ T cell clones. Ligation of the NK cell receptor KIR3DL1 by HLA-Bw4 allotypes resulted in inhibition of cytotoxicity against HLA-B*4403-transfected melanomas as well as against melanomas endogenously expressing HLA-Bw4 allotypes. Similarly, interactions of KIR2DL2 or KIR2DL3 (KIR2DL2/3) with HLA-Cw3-related allotypes on melanomas resulted in decreased tumor cell lysis. We also investigated whether signaling via KIR affected melanoma recognition by CTL. Introduction of KIR3DL1 molecules into HLA-A*0201-restricted gp100-specific CTL resulted in inhibition of lysis of gp100+ melanomas co-expressing HLA-A*0201 and HLA-Bw4 allotypes. These results suggest that disrupting interactions of KIR with their ligands on tumor cells in vivo may enhance antitumor responses mediated by both innate and adaptive immune effector cells.
Perforin and IFN-γ Are Involved in the Antitumor Effects of Antibody-Targeted Superantigens
Authors: Rosendahl Alexander , Kristensson Karin , Hansson Johan , Riesbeck Kristian , Kalland Terje , Dohlsten Mikael
Published: January 2023

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of cytokine production and cytotoxic T cell responses. To target a T cell attack against tumor cells we have genetically engineered a fusion protein of SEA and the Fab part of the tumor-reactive mAb C215. Injection of this Fab-SEA fusion protein to mice carrying lung metastases of the poorly immunogenic B16 melanoma transfected with the C215 Ag resulted in infiltration of cytokine-producing T cells, perforin-containing CTL, and a marked tumor elimination. Fab-SEA therapy induced substantial levels of IFN-γ and TNF-α in serum. In the present study we have characterized the molecular mechanisms of the antitumor effect induced by Fab-SEA treatment in vivo. Neutralization of cytokines by specific Abs demonstrated a major role for IFN-γ in the suppression of tumor growth. In addition, a minor contribution of TNF-α was recorded. Injections of Fab-SEA into normal mice induced strong CTL activity but failed to promote cytotoxic function in perforin knockout mice. Also, a markedly reduced therapy was noted in perforin knockout mice, implicating a role for CTL in Fab-SEA-mediated tumor eradication. The data suggest that Fab-SEA-targeted T cells may suppress tumor growth by both perforin-dependent cytotoxicity and local release of cytokines such as IFN-γ. The latter mechanism may have an important role in cytostatic effects against Ag-negative bystander tumor cells.
1,25-Dihydroxyvitamin D3 Is a Positive Regulator for the Two Anti-Encephalitogenic Cytokines TGF-β1 and IL-4
Authors: Cantorna Margherita T. , Woodward William D. , Hayes Colleen E. , DeLuca Hector F.
Published: January 2023

Previously we demonstrated that 1,25-dihydroxyvitamin D3 blocks the progression of relapsing encephalomyelitis. We now propose that 1,25-dihydroxyvitamin D3 blocks these autoimmune symptoms by stimulating the differentiation and/or function of cells that inhibit the encephalitogenic process. To support this belief, we have found that 1,25-dihydroxyvitamin D3 administration to mice increases IL-4 transcripts by 3- to 25-fold and TGF-β1 transcripts by 4- to 24-fold. Similarly, IL-4 and TGF-β1 transcripts were higher in the central nervous system of 1,25-dihydroxyvitamin D3-treated mice compared with controls. The number of cells recoverable from the lymph nodes of 1,25-dihydroxyvitamin D3-treated mice was only 50% that of controls. Overall, 1,25-dihydroxyvitamin D3 treatment causes a net loss in the total number of lymphocytes while the number of IL-4 and TGF-β1 transcripts increased. The systemic and local increase in the expression of these two anti-inflammatory cytokines by 1,25-dihydroxyvitamin D3 may be responsible for the ability of this drug to block encephalomyelitis.
Fas Ligand-Induced Apoptosis of Infected Human Macrophages Reduces the Viability of IntracellularMycobacterium tuberculosis
Authors: Oddo Mauro , Renno Toufic , Attinger Antoine , Bakker Talitha , MacDonald H. Robson , Meylan Pascal R. A.
Published: January 2023

Mycobacterium tuberculosis-specific cytolytic activity is mediated mostly by CD4+CTL in humans. CD4+CTL kill infected target cells by inducing Fas (APO-1/CD95)-mediated apoptosis. We have examined the effect of Fas ligand (FasL)-induced apoptosis of human macrophages infected in vitro with M. tuberculosis on the viability of the intracellular bacilli. Human macrophages expressed Fas and underwent apoptosis after incubation with soluble recombinant FasL. In macrophages infected either with an attenuated (H37Ra) or with a virulent (H37Rv) strain of M. tuberculosis, the apoptotic death of macrophages was associated with a substantial reduction in bacillary viability. TNF-induced apoptosis of infected macrophages was coupled with a similar reduction in mycobacterial viability, while the induction of nonapoptotic complement-induced cell death had no effect on bacterial viable counts. Infected macrophages also showed a reduced susceptibility to FasL-induced apoptosis correlating with a reduced level of Fas expression. These data suggest that apoptosis of infected macrophages induced through receptors of the TNF family could be an immune effector mechanism not only depriving mycobacteria from their growth environment but also reducing viable bacterial counts by an unknown mechanism. On the other hand, interference by M. tuberculosis with the FasL system might represent an escape mechanism of the bacteria attempting to evade the effect of apoptosis.
Cloning and Characterization of Mouse Vascular Adhesion Protein-1 Reveals a Novel Molecule with Enzymatic Activity
Authors: Bono Petri , Salmi Marko , Smith David J. , Jalkanen Sirpa
Published: January 2023

Human vascular adhesion protein-1 (VAP-1) is a sialylated endothelial cell adhesion molecule mediating the initial L-selectin-independent interactions between lymphocytes and endothelial cells in man. In this work we cloned and characterized mouse VAP-1 (mVAP-1) and produced an anti-mVAP-1 mAb against a recombinant mVAP-1 fusion protein. The isolated cDNA encodes a novel 84.5-kDa mouse molecule. The anti-mVAP-1 mAb stained high endothelial venules in peripheral lymph nodes, and smooth muscle cells and lamina propria vessels in gut. During immunoblotting, this anti-mVAP-1 mAb recognized a 110/220-kDa Ag, suggesting that mVAP-1 is a dimer. Since mVAP-1 has significant sequence identity to members of a family of enzymes called the copper-containing amine oxidases, we showed that mVAP-1 possesses monoamine oxidase activity. Thus, mVAP-1 is the first mouse membrane-bound amine oxidase identified at the molecular level. Based on the 83% identity between the isolated cDNA and human VAP-1 cDNA, the expression pattern, the molecular mass, and the enzyme activity against monoamines, the cloned molecule represents a mouse homologue of human VAP-1. Cloning of mVAP-1 provides a valuable tool for in vivo studies of the significance of VAP-1 for lymphocyte-endothelial cell interactions and of the possible relationship between leukocyte adhesion and amine oxidase activity.
Stromal Cell-Independent Maturation of IL-7-Responsive Pro-B Cells
Authors: Ray Robert J. , Stoddart Angela , Pennycook Jacqueline L. , Huner H. Ozgur , Furlonger Caren , Wu Gillian E. , Paige Christopher J.
Published: January 2023

The proliferation, survival, and differentiation of B cell progenitors in primary hematopoietic tissues depends on extracellular signals produced by stromal cells within the microenvironment. IL-7 is a stromal-derived growth factor that plays a crucial role in B lineage development. We have shown that in the presence of IL-7, pro-B cells proliferate and differentiate to a stage in which they are responsive to stromal cells and LPS, leading to terminally differentiated IgM-secreting plasma cells. In this report, we examine in detail the role of stromal cells in the transition from the IL-7-responsive pro-B cell stage to the mature LPS-responsive B cell stage. We demonstrate that this transition fails to occur, even in the presence of stromal cells and LPS, if constant exposure to IL-7 is maintained. The transition from the large pro-B cell stage to the small cμ+ pre-B cell stage occurs independent of stromal cells. Moreover, the “stromal cell-dependent” maturation that occurs subsequent to the expression of surface IgM leading to responsiveness to B cell mitogens can also be accomplished in the absence of stromal cells if pre-B cells are cultured in proximity to each other or at high cell concentrations. Together these results suggest that stromal cells mediate B cell differentiation by providing the necessary growth requirements (i.e., IL-7) to sustain the development of pre-B cells. The progeny of these pre-B cells can then differentiate through as yet unidentified homotypic interactions, leading to the production of LPS-responsive B cells.
Lack of Both Types 1 and 2 Cytokines, Tissue Inflammatory Responses, and Immune Protection During Pulmonary Infection byMycobacterium bovisBacille Calmette-Guérin in IL-12-Deficient Mice
Authors: Wakeham Julia , Wang Jun , Magram Jeanne , Croitoru Kenneth , Harkness Robin , Dunn Pamela , Zganiacz Anna , Xing Zhou
Published: January 2023

Understanding of key cytokines and the nature of protective immune responses in pulmonary mycobacterial diseases remains a task of paramount importance. In this study, both wild-type (wt) and IL-12-deficient (IL-12−/−) mice were infected by airways inoculation of live Mycobacterium bovis bacille Calmette-Guérin (BCG). The type 1 cytokines IL-12, IFN-γ, and TNF-α, but not the type 2 cytokines IL-4 and granulocyte macrophage (GM)-CSF, markedly increased in the lung and peripheral blood of wt mice postinfection, which resulted in the development of intense granulomatous responses and the effective control of mycobacterial infection in the lung. In contrast, IL-12−/− mice demonstrated a lack of both types 1 and 2 cytokines in the lung and blood and a severely impaired tissue immune-inflammatory response lacking not only macrophages and neutrophils but CD4 and CD8 T cells and NK cells in the lung throughout the entire course of study. Total lung mononuclear cells isolated from these mice, in contrast to wt mice, had an impaired recall immune response to Ag challenge in vitro. These impaired responses resulted in an uncontrolled local growth and systemic spread of bacilli. Our findings reveal that IL-12 plays an irreplaceable role in the initiation of Th1 responses, and the loss of its function cannot be compensated for by alternative mechanisms in the lung. This cytokine, together with IFN-γ and TNF-α, and granulomatous inflammation are critically required for the effective control of pulmonary mycobacterial infection. Our results also indicate that the absence of type 1 cytokines does not necessarily favor a Th2 response.
Humanization and Pharmacokinetics of a Monoclonal Antibody with Specificity for Both E- and P-Selectin
Authors: He Xing-Yue , Xu Zhenghai , Melrose Jennifer , Mullowney Alison , Vasquez Maximiliano , Queen Cary , Vexler Vladimir , Klingbeil Corine , Co Man Sung , Berg Ellen L.
Published: January 2023

E- and P-selectin (CD62E and CD62P) are cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions and are involved in leukocyte recruitment during inflammation. We previously developed a murine mAb, EP-5C7 (or mEP-5C7), that binds and blocks both E- and P-selectin. When used in humans, murine mAbs have short circulating half-lives and generally induce potent human anti-mouse Ab responses. We therefore engineered a humanized, complementarity determining region-grafted version of mEP-5C7 incorporating human γ4 heavy and κ light chain constant regions (HuEP5C7.g4). HuEP5C7.g4 retains the specificity and avidity of mEP-5C7, binding to human E- and P-selectin but not to human L-selectin, and blocking E- and P-selectin-mediated adhesion. Surprisingly, when administered to rhesus monkeys, HuEP5C7.g4 was eliminated from the circulation very rapidly, even faster than the original murine Ab. To isolate the cause of the short serum half-life of HuEP5C7.g4, several Ab variants were constructed. A chimeric IgG4 Ab was made by replacing the humanized V regions with murine V regions. A humanized IgG2 Ab, HuEP5C7.g2, was also made by replacing the human γ4 with a γ2 constant region. Results from pharmacokinetic studies in rhesus monkeys demonstrated that the chimeric IgG4 is also rapidly eliminated rapidly from serum, similar to the humanized IgG4 Ab, while the humanized IgG2 Ab displays a long circulation half-life, typical of human Abs.
Opposite CD4/CD8 Lineage Decisions of CD4+8+ Mouse and Rat Thymocytes to Equivalent Triggering Signals: Correlation with Thymic Expression of a Truncated CD8α Chain in Mice But Not Rats
Authors: Mitnacht Rita , Bischof Astrid , Torres-Nagel Nora , Hünig Thomas
Published: January 2023

Unselected CD4+8+ rat thymocytes, generated in vitro from their direct precursors, are readily converted to functional TCRhigh T cells by stimulation with immobilized TCR-specific mAb plus IL-2. Lineage decision invariably occurs toward CD4−8+, regardless of the timing of TCR stimulation after entry into the CD4+8+ compartment or the concentration of TCR-specific mAb used for stimulation. CD4-specific mAb synergizes with suboptimal TCR-specific mAb in inducing T cell maturation, but lineage decision remains exclusively CD4−8+. These results contrast with those obtained in mice, in which Abs to the TCR complex were shown to promote CD4+8− T cell maturation from CD4+8+ thymocytes. Surprisingly, when rat and mouse CD4+8+ thymocytes were stimulated with PMA/ionomycin under identical conditions, the opposite lineage commitment was observed, i.e., mouse thymocytes responded with the generation of CD4+8− and rat thymocytes with the generation of CD4−8+ cells. It thus seems that CD4+8+ thymocytes of the two species respond with opposite lineage decisions to strong activating signals such as given by TCR-specific mAb or PMA/ionomycin. A possible key to this difference lies in the availability of p56lck for coreceptor-supported signaling. We show that in contrast to mouse CD4+8+ thymocytes, which express both a complete and a truncated CD8α-chain (CD8α′) unable to bind p56lck, rat thymocytes only express full-length CD8α molecules. Mice, but not rats, therefore may use CD8α′ as a “dominant negative” coreceptor chain to attenuate the CD8 signal, thereby facilitating MHC class II recognition through the higher amount of p56lck delivered, and rats may use a different mechanism for MHC class distinction during positive selection.
TGF-β1 Induces the Cyclin-Dependent Kinase Inhibitor p27Kip1 mRNA and Protein in Murine B Cells
Authors: Kamesaki Hiroshi , Nishizawa Kimiko , Michaud Ginette Y. , Cossman Jeffrey , Kiyono Tohru
Published: January 2023

TGF-β1 inhibits the cell cycle progression of many types of cells by arresting them in the G1 phase. This cell cycle arrest has been attributed to the regulatory effects of TGF-β1 on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of proteins, such as p15INK4b, p21WAF1/Cip1, and p27Kip1, that physically associate with cyclins, cyclin-dependent kinases (Cdk), or cyclin-Cdk complexes. In epithelial cell lines, TGF-β1 was previously shown to inhibit cell cycle progression through down-regulation of Cdk4 and/or up-regulation of p15INK4b and/or p21WAF1/Cip1. However, TGF-β1 had little or no effect on the p27Kip1 mRNA and protein levels. In this report, we show that, in contrast to observations in epithelial cell lines, TGF-β1 increased the p27Kip1 mRNA and protein levels in the murine B cell lines CH31 and WEHI231. This TGF-β1-mediated induction of p27Kip1 also resulted in an increased association of p27Kip1 with Cdk2 and a decreased Cdk2 kinase activity. In contrast to epithelial cells, however, TGF-β1 had little or no effect on the Cdk4 and p21WAF1/Cip1 protein levels in these B cells. Finally, although several studies suggested a direct role of p53 in TGF-β1-mediated cell cycle arrest in epithelial cells, TGF-β1 inhibited cell cycle progression in CH31 even in the absence of wild-type p53. Taken together, these results suggest that TGF-β1 induces G1 arrest in B cells primarily through a p53-independent up-regulation of p27Kip1 protein.
Expression of Two Different Antiphagocytic M Proteins byStreptococcus pyogenesof the OF+ Lineage
Authors: Thern Anette , Wästfelt Maria , Lindahl Gunnar
Published: January 2023

All clinical isolates of Streptococcus pyogenes (group A streptococcus) share the ability to resist phagocytosis and grow in human blood. In many strains, this property is due to the expression of a single antiphagocytic M protein, while other strains express more than one M-like molecule, of which the role in phagocytosis resistance is unclear. In particular, all S. pyogenes strains of the OF+ lineage, representing approximately half of all isolates, express two M-like proteins, Mrp and Emm, which are immunologically unrelated. These two proteins bind different ligands that have been implicated in phagocytosis resistance: Mrp binds fibrinogen and Emm binds the complement inhibitor C4BP. Using a clinical isolate of the common serotype 22, we created mutants affected in the mrp and emm genes and characterized them in phagocytosis experiments and by electron microscopy. A double mutant mrp−emm− showed strongly decreased resistance to phagocytosis, while mrp− and emm− single mutants grew well in blood. However, optimal growth required the expression of both Mrp and Emm. Experiments in which coagulation was inhibited using the specific thrombin inhibitor, hirudin, rather than heparin, indicated that Emm is more important than Mrp for resistance to phagocytosis. Tuftlike surface structures typical for S. pyogenes were still present in the mrp−emm− double mutant, but not in a mutant affected in the regulatory gene mga, indicating that the presence of these surface structures is not directly correlated to phagocytosis resistance. Our data imply that OF+ strains of S. pyogenes express two antiphagocytic M proteins with different ligand-binding properties.
Cutting Edge: CD40 Ligand Is Not Essential for the Development of Cell-Mediated Immunity and Resistance toMycobacterium tuberculosis
Authors: Campos-Neto Antonio , Ovendale Pamela , Bement Teresa , Koppi Thelma A. , Fanslow William C. , Rossi Marcos A. , Alderson Mark R.
Published: January 2023

It has been proposed that the induction of cellular immunity and resistance to intracellular pathogens is dependent upon CD40 ligand (CD40L). In the present study we show that this proposal is not ubiquitously supported. Mice genetically deficient in CD40L (CD40LKO) were resistant to i.v. infection with Mycobacterium tuberculosis when assessed by survival and bacteriologic burden in the spleen, liver, and lungs. Infected CD40LKO mice developed granulomas that lacked epithelioid cells and were less numerous and markedly smaller than those observed in control mice. Upon stimulation with purified protein derivative of M. tuberculosis,CD4+ T cells from infected CD40LKO mice proliferated and produced high levels of IFN-γ but not IL-4. Finally, spleen cells from CD40LKO mice stimulated with M. tuberculosis produced IL-12, TNF, and nitric oxide levels comparable to those produced by control cells. In contrast to original proposals, these data clearly show that protective Th1 immunity can be achieved against intracellular pathogens (e.g., Mycobacterium) independently of CD40L.