bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous IL-12 Is Required for Induction and Expression of Experimental Autoimmune Uveitis
Authors: Tarrant Teresa K. , Silver Phyllis B. , Chan Chi-Chao , Wiggert Barbara , Caspi Rachel R.
Published: January 2023

Experimental autoimmune uveitis (EAU) has been associated with a Th1 response. However, in IFN-γ-deficient mice, EAU develops in the context of an effector response having Th2-like elements, and administration of IL-12 to mice immunized for EAU induction can be protective. We, therefore, investigated whether endogenous IL-12 is required for development of EAU. IL-12 p40-deficient mice (12KO) were resistant to EAU induced with the uveitogenic retinal Ag interphotoreceptor retinoid binding protein (IRBP). Delayed hypersensitivity to IRBP was marginally reduced, whereas Ag-specific proliferation was enhanced. Primed lymphocytes of wild-type (wt) mice, cultured with IRBP, produced a Th1-like cytokine profile and transferred EAU to syngeneic wt recipients. Interestingly, the same cells were inefficient in transferring EAU to 12KO recipients, unless IL-12 was included in the culture. Primed cells of the 12KO mice produced a Th2-like cytokine profile and failed to transfer EAU. However, when IL-12 was added to the culture, 12KO cells produced large amounts of IFN-γ and transferred EAU to naive 12KO recipients. We conclude that resistance to EAU of 12KO mice is not due to an inherent inability of these mice to develop ocular disease. Despite an apparent similarity in Ag-specific cytokine responses to IFN-γ-deficient mice, 12KO mice have inhibited generation of uveitogenic effector cells, a situation that can be reversed even after priming, by adding exogenous IL-12 ex vivo. Lastly, the diminished ability of primed wt lymphocytes to induce EAU in 12KO mice indicates a role for endogenous IL-12 in the efferent phase of disease expression that is distinct from its role during Ag priming.
Both PU.1 and Nuclear Factor-κB Mediate Lipopolysaccharide-Induced HIV-1 Long Terminal Repeat Transcription in Macrophages
Authors: Lodie Tracey A. , Reiner Marshall , Coniglio Salvatore , Viglianti Gregory , Fenton Matthew J.
Published: January 2023

We recently reported that LPS stimulation of monocytic cells leads to the activation of PU.1, a member of the Ets family of transcription factors. Phosphorylation of PU.1 by protein kinase CK2 was found to up-regulate its trans-activation function, but not its DNA binding activity. Previous studies suggested that Ets proteins could bind to NF-κB motifs at the tetrameric core sequence TTCC. In macrophages, LPS-inducible HIV-1 gene expression is mediated in part by binding of NF-κB to identical tandem binding sites located within the long terminal repeat (LTR). Thus, we performed additional studies to determine whether PU.1 also played a role in regulating HIV-1 gene expression in macrophages. Our functional studies revealed that activation of the HIV-1 LTR in LPS-stimulated cells requires both NF-κB and PU.1. Extensive mutagenesis of the HIV-1 LTR revealed that PU.1-dependent activation requires the Ets motif within the upstream NF-κB site, whereas NF-κB itself binds to the downstream site. We also found that insertion of five additional nucleotides between the NF-κB sites abolished LPS inducibility, suggesting a direct interaction between factors that bind these sites. Lastly, we found that mutation of PU.1 at serine 148, which prevents its phosphorylation by CK2, blocked its ability to activate the HIV-1 LTR in response to LPS. These effects were promoter specific because PU.1 did not affect LPS-inducible activation of a distinct NF-κB-dependent promoter. While these data do not demonstrate direct binding of PU.1 to the HIV-1 LTR, they illustrate a novel role for PU.1 in activation of the HIV-1 LTR by LPS.
Crucial Role of TCRγ Chain Junctional Region in Prenyl Pyrophosphate Antigen Recognition by γδ T Cells
Authors: Bukowski Jack F. , Morita Craig T. , Band Hamid , Brenner Michael B.
Published: January 2023

Human γδ T cells recognize prenyl pyrophosphate Ags and their analogues in a Vγ2Vδ2 TCR-dependent manner. Few data are available regarding the TCR structural requirements for recognition of such prenyl pyrophosphate Ags by γδ T cells. Presently, we made chain pair switch, chimeric, and site mutant γδ TCRs and transfected them into TCR− mutant Jurkat T cells to examine the effects of changing the TCRγ junctional region sequences on reactivity to prenyl pyrophosphate Ags. Substitution of the TCRγ junctional region (N and J) sequences from an Ag-reactive TCR with TCRγ junctional region sequences from an Ag-nonreactive TCR abrogated reactivity to the prenyl pyrophosphate Ag isopentenyl pyrophosphate and to its synthetic analogue ethyl pyrophosphate but not to a mycobacterial supernatant containing a mixture of prenyl pyrophosphate Ags. Substitution of only the TCRγ N nucleotide region with that from this Ag-nonreactive TCR destroyed reactivity to isopentenyl pyrophosphate and to the mycobacterial supernatant. Substitution of the entire Vδ2 chain from the Ag-reactive TCR with a Vδ1 chain from an Ag-nonreactive TCR yielded a prenyl pyrophosphate Ag-nonreactive TCR. Thus, using TCR mutagenesis and TCR transfectants, we show that γδ TCR reactivity to prenyl pyrophosphate Ags is dependent upon the junctional region of the TCRγ chain and upon pairing of Vγ2 and Vδ2 TCR chains. These structural requirements of TCRγδ recognition of prenyl pyrophosphates distinguish this reactivity from that of protein superantigens and emphasize the importance of the TCRγ CDR3 loop and adjacent residues.
A λ 3′ Enhancer Drives Active and Untemplated Somatic Hypermutation of a λ1 Transgene
Authors: Kong Qingzhong , Zhao Lisa , Subbaiah Sathish , Maizels Nancy
Published: January 2023

Somatic hypermutation is a highly regulated process that targets mutations to the rearranged Ig genes. Little is known about the cis-elements required for somatic hypermutation of the λ light chain gene. We have studied somatic hypermutation of a rearranged λ1 transgene under the control of either a λ2-4 or κ 3′ enhancer. The mutations in the transgenes were analyzed by sequencing DNA amplified from hypermutating Peyer’s patch B cells. The results indicate that the λ 3′ enhancer can drive active hypermutation of a λ1 transgene in Peyer’s patch cells. The λ1 transgene under analysis carried two marked Vλ2 genes immediately upstream that could serve as sequence donors in possible gene conversion events. There was no evidence of sequence transfer to the hypermutated λ1 gene, suggesting that gene conversion is not a major mechanism for somatic hypermutation in mice.
Susceptibility to Pneumocystis carinii in Mice Is Dependent on Simultaneous Deletion of IFN-γ and Type 1 and 2 TNF Receptor Genes
Authors: Rudmann Daniel G. , Preston Angela M. , Moore Mark W. , Beck James M.
Published: January 2023

Pneumocystis carinii pneumonia is an important cause of morbidity and mortality in immunosuppressed patients, particularly HIV-infected individuals. An improved understanding of pulmonary host response, including the cytokines required for defense, could suggest novel immunotherapeutic approaches to this infection. The cytokines IFN-γ and TNF have contributory roles in host defense against P. carinii, but their combined and interactive importance is unclear. To test the roles of these cytokines in defense against P. carinii directly, organisms were inoculated intratracheally into wild-type mice and into three groups of gene-deleted mice: those lacking genes for IFN-γ (IFN-γ−/−), for TNF receptors 1 and 2 (TNFR−/−), and for both IFN-γ and TNFR (TNFR-IFN-γ−/−). Four weeks after P. carinii inoculation, lungs of the wild-type, IFN-γ−/−, and TNFR−/− mice demonstrated clearance of P. carinii and only mild inflammation. However, TNFR-IFN-γ−/− mice demonstrated severe P. carinii infection and lung inflammation. Our findings demonstrate conclusively that deletion of either IFN-γ or TNF activity alone does not block clearance of P. carinii. However, simultaneous deletion of IFN-γ and TNF receptor genes results in susceptibility to P. carinii. Rather than focusing exclusively on individual cytokines, our data suggest that immunotherapy targeted at maximizing both the IFN-γ and TNF responses to P. carinii may be required to augment host defense against this important opportunistic pathogen.
Differential Expression and Function of L-Selectin on CD56bright and CD56dim Natural Killer Cell Subsets
Authors: Frey Margaret , Packianathan Nalini B. , Fehniger Todd A. , Ross Mary E. , Wang Wan-Chao , Stewart Carleton C. , Caligiuri Michael A. , Evans Sharon S.
Published: January 2023

NK cells are the first line of defense against foreign cells, virally infected cells, and tumors. The mechanisms whereby NK cells accumulate in extralymphoid sites in response to pathogenic stimuli are not well understood. The L-selectin adhesion molecule (CD62L) plays a primary role in mediating the initial interaction of leukocytes with vascular endothelium, a crucial step in the extravasation of immune effector cells into tissues. In this report, we show L-selectin to be uniquely expressed on a subset of resting human NK cells (CD56bright). Notably, CD56bright NK cells expressed L-selectin at a higher density than all other peripheral blood leukocytes. NK activation by PMA, IL-2, IL-15, or TGF-β down-regulated L-selectin on the CD56bright subset, while increased L-selectin levels were observed in both the CD56bright and CD56dim NK subsets in response to IL-12, IL-10, or IFN-α. Moreover, CD56bright NK cells bound with high efficiency to physiologic L-selectin ligands on peripheral lymph node high endothelial venules (HEV). In sharp contrast, CD56dim NK cells adhered poorly to HEV and were predominantly L-selectin− or expressed L-selectin only at low density. In CD56bright cells and a subpopulation of CD56dim cells, L-selectin ligation by mAb cross-linking activated lymphocyte function-associated Ag 1 (LFA-1), a second adhesion molecule required for leukocyte extravasation. LFA-1 was expressed on both NK subsets, although its density was constitutively higher on CD56dim cells. Taken together, evidence of differential expression of L-selectin and LFA-1 on CD56bright and CD56dim NK subsets strongly suggests unique migratory properties and functions of these cells during the early immune response to foreign pathogens.
Oral Administration of the Bacterial Superantigen Staphylococcal Enterotoxin B Induces Activation and Cytokine Production by T Cells in Murine Gut-Associated Lymphoid Tissue
Authors: Spiekermann Gerburg M. , Nagler-Anderson Cathryn
Published: January 2023

The toxicity of the staphylococcal enterotoxins (SEs) has been linked to the activation of large numbers of T cells in the peripheral lymphoid tissues. Because the primary manifestations of foodborne enterotoxic poisoning are associated with the gastrointestinal tract, we have compared the responses of T cells in the gut-associated lymphoid tissue and in the periphery to intragastric (i.g.) and i.p. administration of SEB. Intraperitoneal SEB results in an early expansion of peripheral Vβ8+ T cells and Th1 cytokine secretion followed by deletion at 7–10 days. We found that i.g. SEB rapidly (within 4 h) leads to the expansion and activation of Vβ8+ T cells in the Peyer’s patch and mesenteric lymph nodes. Analysis of cytokine mRNA in purified Vβ8+ T cells by competitive RT-PCR showed that, 4 h after i.g. SEB, the induction of mRNA for IL-2 and IFN-γ is about 10-fold greater in mucosal than in peripheral lymphoid tissue. Our results show that activated mucosal T cells expand and up-regulate cytokine mRNA in response to luminal exposure to SEB, suggesting a role for the gut-associated lymphoid tissue in the gastrointestinal manifestations of enterotoxic poisoning.
Astrocytes Express Elements of the Class II Endocytic Pathway and Process Central Nervous System Autoantigen for Presentation to Encephalitogenic T Cells
Authors: Soos Jeanne M. , Morrow Jennifer , Ashley Timothy A. , Szente Brian E. , Bikoff Elizabeth K. , Zamvil Scott S.
Published: January 2023

Astrocytes are nonprofessional APCs that may participate in Ag presentation and activation of pathogenic CD4+ T cells involved in central nervous system (CNS) inflammatory diseases. Using immortalized pure astrocytes as a complement to the study of primary astrocytes, we investigated whether these astrocytes express elements involved in the class II endocytic pathway and if they are capable of processing native myelin basic protein (MBP), a step that could be necessary for initiating or perpetuating T cell recognition of this self-Ag in vivo. Upon IFN-γ-stimulation, primary and immortalized astrocytes up-regulate class II transactivator (CIITA), invariant chain (Ii) (p31 and p41), H-2Ma, and H-2Mb. Analysis of CIITA cDNA sequences demonstrated that CIITA transcription in astrocytes is directed by a promoter (type IV) that mediates IFN-γ-inducible CIITA expression and encodes a CIITA protein that differs in its N-terminal sequence from CIITA reported in professional APC. Comparing live and fixed APC for Ag presentation, we show that Ag processing by APC is required for presentation of native MBP to autopathogenic T cells specific for the major MBP epitope, Ac1-11. We have observed that primary astrocytes and some, but not all, astrocyte lines in the absence of contaminating microglia are capable of processing and presenting native MBP, suggesting that there may be heterogeneity. Our study provides definitive evidence that astrocytes are capable of processing CNS autoantigen, indicating that astrocytes have potential for processing and presentation of CNS autoantigen to proinflammatory T cells in CNS autoimmune disease.
External and Internal Calibration of the MHC Class I-Specific Receptor Ly49A on Murine Natural Killer Cells
Authors: Kåse Anna , Johansson Maria H. , Olsson-Alheim Mats Y. , Kärre Klas , Höglund Petter
Published: January 2023

Expression of the H-2Dd-specific inhibitory receptor Ly49A on murine NK cells is subject to MHC class I-dependent modulation in vivo. As a result, NK cells in H-2Dd-transgenic mice express low cell surface levels of Ly49A, whereas NK cells from nontransgenic C57BL/6 (B6) mice express high levels. The purpose of this study was to assess the role of MHC class I molecules on the NK cell itself vs those on surrounding cells in this calibration and to test whether the Ly49A levels are subject to regulation in mature NK cells also. Analysis of transgenic mice with mosaic expression of an H-2Dd/Ld transgene showed that MHC class I molecules on surrounding cells (external ligands) and on the NK cell itself (internal ligands) played distinct roles in the determination of Ly49A levels. External ligands were involved in down-regulation of Ly49A levels in vivo, whereas internal ligands kept the down-regulated levels of Ly49A low upon NK cell activation in vitro. Furthermore, in an experimental system based on adoptive transfer of spleen cells, receptor down-regulation of Ly49A occurred as a rapid adaptation process in mature NK cells after interaction with the H-2Dd ligand in vivo. This suggests that Ly49 levels are not fixed but can be changed in mature NK cells when they are exposed to a changed MHC class I environment.
The IL-4 Rapidly Produced in BALB/c Mice After Infection with Leishmania major Down-Regulates IL-12 Receptor β2-Chain Expression on CD4+ T Cells Resulting in a State of Unresponsiveness to IL-12
Authors: Himmelrich Hayo , Parra-Lopez Carlos , Tacchini-Cottier Fabienne , Louis Jacques A. , Launois Pascal
Published: January 2023

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R β2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R β2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R β2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R β2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA β2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-γ, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R β2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.
FLT3-Ligand Administration Inhibits Liver Metastases: Role of NK Cells
Authors: Péron Jean-Marie , Esche Clemens , Subbotin Vladimir M. , Maliszewski Charles , Lotze Michael T. , Shurin Michael R.
Published: January 2023

FLT3-ligand (FL) is a recently described cytokine that stimulates the proliferation and differentiation of hematopoietic progenitors both in vivo and in vitro and, when administered to mice, induces an accumulation of dendritic cells (DC) in different lymphoid and nonlymphoid organs and tissues, including the liver. We have studied the antitumor effect of FL administered alone or in combination with IL-12 in a day 3 murine liver metastasis model. FL significantly reduced the number of hepatic metastases (36.00 ± 11.00 vs 92.00 ± 10.19 in control group, p < 0.05). Histologic evaluation of the livers revealed that FL induced a significant infiltration of the tumor border by lymphocytes and DC associated with increased number of apoptotic figures. Immunohistochemical analysis demonstrated that FL significantly enhanced the number of DC in the liver parenchyma and within the liver metastases, as well as the number of CD4+ and CD8+ T lymphocytes. These data support the suggestion that DC may be directly involved in the antitumor effect of FL. Interestingly, the antitumor effect of FL was greatly reduced by the NK depletion. Combination of FL and IL-12 resulted in greater antitumor efficacy than these cytokines alone. In summary, we have shown that FL has significant antitumor effect on preexisting murine C3 liver tumors that is mediated by NK cells. We have also demonstrated that the FL/IL-12 combination has an enhanced antitumor activity in the same murine tumor model.
Interaction of the Fungal PathogenCandida albicanswith Integrin CD11b/CD18: Recognition by the I Domain Is Modulated by the Lectin-Like Domain and the CD18 Subunit
Authors: Forsyth Christopher B. , Plow Edward F. , Zhang Li
Published: January 2023

Interactions of microorganisms with integrins are central to the host defense mechanisms. The leukocyte integrin CD11b/CD18 is the principal adhesion receptor on leukocytes for Candida albicans, a major opportunistic pathogen. In this study we have investigated the roles of three regions within the receptor, the inserted (I) and lectin-like domains within the CD11b subunit, and the CD18 subunit, in CD11b/CD18-C. albicans interactions. We report four major findings. 1) A mutation in CD18 exerts a dominant negative effect on the function of the CD11b/CD18 complex. This interpretation is based on the observation that in the absence of CD18, the CD11b subunit alone binds C. albicans well, but a single point mutation at Ser138 of CD18 abolishes CD11b/CD18 binding of the fungus. 2) The lectin-like domain is not sufficient for CD11b/CD18-C. albicans interactions. Rather, the lectin-like domain appears to influence CD11b/CD18 binding activity by modulating the function of the I domain. 3) The I domain is the primary binding site for C. albicans in the receptor and is sufficient to support an efficient interaction. 4) We have identified specific amino acid sequences within the I domain that engage the microorganism. Compared with other ligands of CD11b/CD18, C. albicans has some unique as well as common contact sites within the I domain of the receptor. Such unique contact sites may underlie the ability of C. albicans to modulate CD11b/CD18 function and raise the possibility for selective interference of the microorganism-host leukocyte interactions.
Host Genetic Determinants of Vaccine-Induced Eosinophilia During Respiratory Syncytial Virus Infection
Authors: Hussell Tracy , Georgiou Andrew , Sparer Tim E. , Matthews Stephen , Pala Pietro , Openshaw Peter J. M.
Published: January 2023

In BALB/c mice, sensitization with the attachment protein (G) of respiratory syncytial virus (RSV) leads to CD4+ T cell-mediated lung eosinophilia during subsequent challenge with RSV. To determine the host genetic influences on this model of lung eosinophilia, we tested 15 different inbred mouse strains. Eosinophilia developed in all H-2d (BALB/c, DBA/2n, and B10.D2), but not in H-2k (CBA/Ca, CBA/J, C3H, BALB.K, or B10.BR) mouse strains. Among H-2b mice, 129 and BALB.B developed eosinophilia, whereas C57BL/6 and C57BL/10 did not. Testing first generation crosses between sensitive and resistant strains showed that eosinophilia developed in all H-2dxk (n = 5), irrespective of background genes, but not in H-2dxb (n = 2) mice. In vivo depletion of CD8+ T cells or IFN-γ rendered C57BL/6, but not BALB.K mice, susceptible to eosinophilia. Analysis of B10 recombinant mice showed that the Dd allele (in B10.A(5R) mice) prevented CD8+ T cell accumulation in the lung, resulting in intense lung eosinophilia. However, the Db allele (in B10.A(2R) and B10.A(4R) mice) supported CD8+ T cell expansion and prevented eosinophilia. Intracellular cytokine staining showed that lung eosinophilia correlated with reduced IFN-γ and increased IL-10 expression in lung T cells. These results are compatible with the unifying model that Th2 cells mediate the disease but can be inhibited by CD8+ T cells secreting IFN-γ. Our findings have important implications for the development of protective, nonpathogenic vaccines for RSV disease.
α(1,3)-Fucosyltransferase VII-Dependent Synthesis of P- and E-Selectin Ligands on Cultured T Lymphoblasts
Authors: Knibbs Randall N. , Craig Ronald A. , Mály Petr , Smith Peter L. , Wolber Frances M. , Faulkner Neil E. , Lowe John B. , Stoolman Lloyd M.
Published: January 2023

T lymphocytes up-regulate the synthesis of ligands for E- and P-selectin during proliferative responses in vivo and in vitro. Previous studies from our laboratories indicated that the α(1,3)-fucosyltransferase FucT-VII regulates the synthesis of E-selectin ligands and sialylated Lewisx-related epitopes (sLex-related epitopes) in human T lymphoblasts. The current report shows that production of both P- and E-selectin ligands is FucT-VII dependent, but peak synthesis of each occurs at different levels of fucosyltransferase activity in intact cells. In brief, FucT-VII mRNA levels were higher in cultured T lymphoblasts expressing sLex-related epitopes and both selectin ligands than in cells expressing P-selectin ligands alone. However, synthesis of the epitopes and both selectin ligands required the FucT-VII enzyme in transfected Molt-4 cells. In contrast, neither constitutive nor transfection-enhanced levels of the FucT-IV enzyme generated active P-selectin ligands in these lines. In addition, targeted deletion of the FucT-VII gene in mice markedly inhibited the synthesis of both P- and E-selectin ligands during blast transformation in vitro. Finally, the optimal synthesis of active P-selectin ligands occurred at lower level of FucT-VII activity than required for synthesis of equally active E-selectin ligands in both cultured T lymphoblasts and FucT-VII transfectants. Consequently, the FucT-VII enzyme is essential for the synthesis of both P- and E-selectin ligands by T lymphoblasts, and its activity determines whether P-selectin ligands are expressed alone or in conjunction with E-selectin ligands and sLex-related epitopes on human T cells.
The Selective Inhibition of β1 and β7 Integrin-Mediated Lymphocyte Adhesion by Bacitracin
Authors: Mou Yanglong , Ni Heyu , Wilkins John A.
Published: January 2023

Integrins play an important role in lymphocyte adhesion to cellular and extracellular components of their microenvironment. The regulation of such adhesion often involves changes in the functional state of the integrins rather than alterations in their expression levels. Although the functional basis for such transitions is unknown, a possible role for disulfide exchange might be postulated based on the observations that integrin function can be activated by bifunctional reducing agents or by Abs that react with areas adjacent to predicted long-range disulfide bonds in integrins. Recently, it has been reported that enzymes that catalyze disulfide exchanges such as protein disulfide isomerase (PDI) are present on the surface of lymphoid cells, raising the possibility that such enzymes might be involved in the control of lymphocyte adhesion. A number of inhibitors of PDI function were examined for their effects on integrin-mediated adherence of T cells. The results did not support role for PDI in the regulation of integrin function, as the inhibitors somatostatin A, tocinoic acid, dithiobisnitrobenzoic acid, and anti-PDI mAb did not interfere with adherence. However, one of the PDI inhibitors, bacitracin, selectively interfered with the β1 integrin-mediated adherence of lymphoid cells to collagen, fibronectin, laminin, and VCAM-1, and with α4β7-dependent adherence to fibronectin and to VCAM-1. In contrast, αvβ3- and αLβ2-mediated adherence were not inhibited. Thus, it appears that bacitracin may be a selective inhibitor of β1 and β7 integrin functions by an as yet unknown mechanism.
Dengue Virus Infection of Human Endothelial Cells Leads to Chemokine Production, Complement Activation, and Apoptosis
Authors: Avirutnan Panisadee , Malasit Prida , Seliger Barbara , Bhakdi Sucharit , Husmann Matthias
Published: January 2023

Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe complications of secondary dengue virus (DV) infection. Vascular leakage, hemorrhagic diathesis and complement activation are the hallmarks of the disease. The short-lived nature of the plasma leakage syndrome has led to the conclusion that altered permeability is most likely effected by a soluble mediator. In the present study, we show that infection of human endothelial cells with DV induces the transcriptional up-regulation and secretion of RANTES and IL-8 and, in the presence of anti-dengue Abs, the formation of nonlytic complement complexes. Extremely high levels of IL-8 were detected in plasma and pleural fluid samples from patients with DSS. Furthermore, DV infection of endothelial cells in vitro caused apoptosis. Complement activation, chemokine induction, and apoptotic cell death may act in concert to cause the fulminant but short-lived vascular leakage that is characteristic of DHF/DSS.
Macrophage Colony-Stimulating Factor Stimulates Synthesis and Secretion of a Mouse Homolog of a Human IgE-Dependent Histamine-Releasing Factor by Macrophages In Vitro and In Vivo
Authors: Teshima Shigetada , Rokutan Kazuhito , Nikawa Takeshi , Kishi Kyoichi
Published: January 2023

Treatment of murine resident peritoneal macrophages with macrophage-CSF (M-CSF) up-regulated the synthesis of a discrete set of proteins, including a 26-kDa protein (p26). The sequence of 20 NH2-terminal amino acids of the purified p26 was identical with the mouse homolog of a human IgE-dependent histamine-releasing factor (HRF). Among macrophage activators tested (M-CSF, granulocyte-macrophage-CSF, IL-3, TNF-α, IFN-γ, and LPS), only M-CSF could up-regulate the p26 HRF synthesis by cultured macrophages. M-CSF not only increased the levels of p26 HRF mRNA and protein, but also stimulated the secretion of an N-glycosylated p26 HRF with a m.w. of 30 kDa. Repeated injections of M-CSF into mouse peritoneal cavity for 4 days elicited macrophages expressing abundant p26 HRF. A single i.p. injection of M-CSF failed to increase the p26 HRF level in peritoneal macrophages of thioglycollate-, LPS-, or adjuvant-treated mice, while M-CSF challenge to OVA-immunized mice caused macrophage infiltration and overproduction of p26 HRF, similarly as did OVA challenge. The Ag-specific priming for enhanced synthesis and secretion of p26 HRF by M-CSF was also demonstrated in cultured macrophages prepared from OVA-immunized mice. An i.p. injection of M-CSF or recombinant p26 HRF triggered eosinophil recruitment, even in the absence of the Ag, in the sensitized mice, but not in normal mice. Furthermore, recombinant p26 HRF could induce eosinophilia without marked macrophage and lymphocyte infiltrations. Our results suggest that p26 HRF secreted by M-CSF-stimulated macrophages may be an important mediator for the late phase allergic inflammation.
The Role of CD8+ CD40L+ T Cells in the Formation of Germinal Centers in Rheumatoid Synovitis
Authors: Wagner Ulf G. , Kurtin Paul J. , Wahner Andrea , Brackertz Marc , Berry Daniel J. , Goronzy Jörg J. , Weyand Cornelia M.
Published: January 2023

In rheumatoid synovitis, lymphocytes can be arranged in follicular structures resembling secondary lymphoid follicles. To understand the organizing principles of this ectopic lymphoid tissue, the cellular components contributing to synovial follicles were examined. In 9 of 24 synovial tissue biopsies, lymphoid aggregates were found consisting of CD4+ T cells and CD20+ B cells. In four of the nine patients, the follicular centers were occupied by CD23+ CD21+ cellular networks representing follicular dendritic cells involved in germinal center reactions. In five patients, CD23+ cells were absent from the centers of the aggregates, suggesting that fully developed germinal centers are generated in only a subset of patients. To identify factors involved in the regulation of the synovial microarchitecture, cell populations contributing to the follicles were quantified by digital image analysis of immunostained tissue and by flow cytometry of tissue-derived lymphocytes. Proportions of CD4+, CD20+, and CD68+ cell subsets were surprisingly invariant, irrespective of the presence or absence of CD23+ follicular dendritic cells. Instead, tissue biopsies with CD23+ germinal center-like regions could be distinguished from those with CD23− T cell-B cell aggregates by a fourfold increase in the frequency of tissue-infiltrating CD8+ T cells, a fraction of which expressed CD40 ligand (CD40L). The data suggest a previously unsuspected role of CD8+ lymphocytes in modulating germinal center formation and raise the possibility that CD8+ CD40L+ T cells are involved in aggravating pathologic immune responses in rheumatoid synovitis.
Proteolytic Cleavage of ICAM-1 by Human Neutrophil Elastase
Authors: Champagne Benoit , Tremblay Pierre , Cantin André , St. Pierre Yves
Published: January 2023

Human leukocyte elastase (HLE) participates in tissue destruction in a number of inflammatory disorders, including rheumatoid arthritis and cystic fibrosis. Since HLE has been shown to bind to Mac-1, and ICAM-1 plays a key role during the recruitment and the activation of leukocytes at inflamed sites, we investigated the capacity of HLE to cleave ICAM-1. Flow-cytometric analyses showed a dose-dependent cleavage of ICAM-1 by HLE on different human cell lines. The cleavage was completely inhibited by α1-antitrypsin, a natural HLE protease inhibitor. The ability of HLE to degrade ICAM-1 was further confirmed by electrophoretic analysis using a soluble form of ICAM-1 (D1-D5). Enzymatic removal of N-linked glycosylation did not significantly modulate ICAM-1 cleavage by HLE, while removal of sialic acid residues partially reduced the sensitivity of ICAM-1 to HLE. We further showed that sputum of cystic fibrosis patients contains high levels of HLE activity capable of cleavage of cell surface ICAM-1. The cleavage induced by incubation of cells with the sputum sample was totally inhibited by α1-antitrypsin and the specific peptidic HLE inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone. Moreover, the cleavage of ICAM-1 was concomitant to that of CD4 at the surface of the same cell, at the same amplitude, and at all HLE concentrations. The capacity of HLE to modulate the expression of ICAM-1 on the surface of leukocytes by proteolytic cleavage brings support to the hypothesis that overproduction of HLE can cause severe immunologic lung disorders by affecting intercellular adhesion.
Central T Cell Tolerance in Lupus-Prone Mice: Influence of Autoimmune Background and the lpr Mutation
Authors: Fatenejad Saeed , Peng Stanford L. , Disorbo Olimpia , Craft Joe
Published: January 2023

Lupus-prone mice develop a systemic autoimmune disease that is dependent upon the B cell help provided by autoreactive αβ CD4+ T cells. Since autoreactive T cells with high affinity for self peptides are normally deleted in the thymus, their presence in these mice suggests the possibility that intrathymic negative selection may be defective. Here, we directly compared central T cell tolerance in response to a conventional peptide Ag in lupus-prone MRL/MpJ mice with a nonautoimmune strain using an MHC class II-restricted TCR transgene. Our results did not demonstrate any defects after Ag exposure in the induction of intrathymic deletion of immature CD4+CD8+ thymocytes, in TCR down-regulation, or in the number of apoptotic thymocytes in MRL/MpJ compared with nonautoimmune mice. Furthermore, we found that the lpr mutation had no influence upon the Ag-induced thymic deletion of immature thymocytes. These data support the notion that T cell autoreactivity in MRL/MpJ mice is caused by defects in peripheral control mechanisms.
Natural Killer Cells from HIV-1+ Patients Produce C-C Chemokines and Inhibit HIV-1 Infection
Authors: Fehniger Todd A. , Herbein Georges , Yu Haixin , Para Michael I. , Bernstein Zale P. , O’Brien William A. , Caligiuri Michael A.
Published: January 2023

Human NK cells have been shown to produce cytokines (e.g., IFN-γ and TNF-α) and the chemokine macrophage inflammatory protein (MIP)-1α following stimulation with the combination of two monokines, IL-15 plus IL-12. The C-C chemokines MIP-1α, MIP-1β, and RANTES have been identified as the major soluble macrophage-tropic HIV-1-suppressive factors produced by CD8+ T cells, which exert their action at the level of viral entry. Here, we demonstrate that monokine-activated NK cells, isolated from both normal and HIV-1+ donors, produce similar amounts of MIP-1α, MIP-1β, and RANTES protein, in vitro. Further, supernatants of monokine-activated NK cells obtained from both normal donors and AIDS patients showed potent (routinely ≥90%) suppressive activity against HIV-1 replication in vitro, compared with unstimulated control supernatants. NK cell supernatants inhibited both macrophage-tropic HIV-1NFN-SX and T cell-tropic HIV-1NL4–3 replication in vitro, but not dual-tropic HIV-189.6. Importantly, the C-C chemokines MIP-1α, MIP-1β, and RANTES were responsible only for a fraction of the HIV-1-suppressive activity exhibited by NK cell supernatants against macrophage-tropic HIV-1. Collectively these data indicate that NK cells from normal and HIV-1+ donors produce C-C chemokines and other unidentified factors that can inhibit both macrophage- and T cell-tropic HIV-1 replication in vitro. Since NK cells can be expanded in patients with HIV-1, AIDS, and AIDS malignancy in vivo, this cell type may have an important role in the in vivo regulation of HIV-1 infection.
Cutting Edge: Functional Characterization of the Effect of the C3H/HeJ Defect in Mice that Lack an Lps
n Gene: In Vivo Evidence for a Dominant Negative Mutation
Authors: Vogel Stefanie N. , Johnson Dabney , Perera Pin-Yu , Medvedev Andrei , Larivière Line , Qureshi Salman T. , Malo Danielle
Published: January 2023

A point mutation in the Tlr4 gene, which encodes Toll-like receptor 4, has recently been proposed to underlie LPS hyporesponsiveness in C3H/HeJ mice (Lpsd). The data presented herein demonstrate that F1 progeny from crosses between mice that carry a ∼9-cM deletion of chromosome 4 (including deletion of LpsTlr4) and C3H/HeJ mice (i.e., Lps0 × Lpsd F1 mice) exhibit a pattern of LPS sensitivity, measured by TNF activity, that is indistinguishable from that exhibited by Lpsn × Lpsd F1 progeny and whose average response is “intermediate” to parental responses. Thus, these data provide clear functional support for the hypothesis that the C3H/HeJ defect exerts a dominant negative effect on LPS sensitivity; however, expression of a normal Toll-like receptor 4 molecule is apparently not required.
Cutting Edge: Identification of the Orphan Chemokine Receptor GPR-9-6 as CCR9, the Receptor for the Chemokine TECK
Authors: Zaballos Ángel , Gutiérrez Julio , Varona Rosa , Ardavín Carlos , Márquez Gabriel
Published: January 2023

Thymus-expressed chemokine (TECK) has been reported to chemoattract dendritic cells, thymocytes, and activated macrophages. Here, we show that TECK is a specific agonist for a human orphan receptor called GPR-9-6. We have determined the cDNA sequence of human GPR-9-6 and cloned the corresponding murine cDNA. Human and murine GPR-9-6 expression is very high in the thymus and low in lymph nodes and spleen. RT-PCR analysis of murine GPR-9-6 expression on murine FACS-sorted thymocyte subpopulations showed that this gene is expressed in both immature and mature T cells. Additions of human or murine TECK to HEK 293/human GPR-9-6 and HEK 293/murine GPR-9-6 transfectants provoked intracytoplasmic calcium mobilization. Human TECK also induced the in vitro migration of HEK 293/human GPR-9-6 cells. These results confirm that GPR-9-6 is a specific receptor for TECK. According to the established nomenclature system, we propose to rename GPR-9-6 as CC chemokine receptor 9 (CCR9).
Cutting Edge: C1q Protects Against the Development of Glomerulonephritis Independently of C3 Activation
Authors: Mitchell Daniel A. , Taylor Philip R. , Cook H. Terence , Moss Jill , Bygrave Anne E. , Walport Mark J. , Botto Marina
Published: January 2023

C1q-deficient (C1qa−/−) mice develop antinuclear Abs and glomerulonephritis (GN) characterized by multiple apoptotic bodies. To explore the contribution of C3 activation to the induction of spontaneous GN, C1qa−/− mice were crossed with factor B- and C2-deficient (H2-Bf/C2−/−) mice. GN was present in 64% of the 45 C1qa/H2-Bf/C2−/− mice compared with 8% of the 65 H2-Bf/C2−/− mice and none of the 24 wild-type controls. IgG was detected in the glomeruli of diseased C1qa/H2-Bf/C2−/− kidneys. However, glomerular staining for C3 was absent. Increased numbers of glomerular apoptotic bodies were detected in undiseased C1qa/H2-Bf/C2−/− kidneys. These findings support the hypothesis that C1q may play a role in the clearance of apoptotic cells without the necessity for C3 activation and demonstrate that the activation of C3 is not essential for the development of GN in this spontaneous model of lupus-like disease.
Unopposed Production of Granulocyte-Macrophage Colony-Stimulating Factor by Tumors Inhibits CD8+ T Cell Responses by Dysregulating Antigen-Presenting Cell Maturation
Authors: Bronte Vincenzo , Chappell Dale B. , Apolloni Elisa , Cabrelle Anna , Wang Michael , Hwu Patrick , Restifo Nicholas P.
Published: January 2023

Tumor cells gene-modified to produce GM-CSF potently stimulate antitumor immune responses, in part, by causing the growth and differentiation of dendritic cells (DC). However, GM-CSF-modified tumor cells must be γ-irradiated or they will grow progressively, killing the host. We observed that 23 of 75 (31%) human tumor lines and two commonly used mouse tumor lines spontaneously produced GM-CSF. In mice, chronic GM-CSF production by tumors suppressed Ag-specific CD8+ T cell responses. Interestingly, an inhibitory population of adherent CD11b(Mac-1)/Gr-1 double-positive cells caused the observed impairment of CD8+ T cell function upon direct cell-to-cell contact. The inhibitory cells were positive for some markers associated with Ag presenting cells, like F4/80, but were negative for markers associated with fully mature DC like DEC205, B7.2, and MHC class II. We have previously reported that a similar or identical population of inhibitory “immature” APC was elicited after immunization with powerful recombinant immunogens. We show here that these inhibitory cells can be elicited by the administration of recombinant GM-CSF alone, and, furthermore, that they can be differentiated ex vivo into “mature” APC by the addition of IL-4 and GM-CSF. Thus, tumors may be able to escape from immune detection by producing “unopposed” GM-CSF, thereby disrupting the balance of cytokines needed for the maturation of fully functional DC. Further, CD11b/Gr-1 double-positive cells may function as “inhibitory” APC under the influence of GM-CSF alone.
Antigen-Specific Therapy of Murine Lupus Nephritis Using Nucleosomal Peptides: Tolerance Spreading Impairs Pathogenic Function of Autoimmune T and B Cells
Authors: Kaliyaperumal Arunan , Michaels Marissa A. , Datta Syamal K.
Published: January 2023

In the (SWR × NZB)F1 mouse model of lupus, we previously localized the critical autoepitopes for nephritogenic autoantibody-inducing Th cells in the core histones of nucleosomes at aa positions 10–33 of H2B and 16–39 and 71–94 of H4. A brief therapy with the peptides administered i.v. to 3-mo-old prenephritic (SWR × NZB)F1 mice that were already producing pathogenic autoantibodies markedly delayed the onset of severe lupus nephritis. Strikingly, chronic therapy with the peptides injected into 18-mo-old (SWR × NZB)F1 mice with established glomerulonephritis prolonged survival and even halted the progression of renal disease. Remarkably, tolerization with any one of the nucleosomal peptides impaired autoimmune T cell help, inhibiting the production of multiple pathogenic autoantibodies. However, cytokine production or proliferative responses to the peptides were not grossly changed by the therapy. Moreover, suppressor T cells were not detected in the treated mice. Most interestingly, the best therapeutic effect was obtained with nucleosomal peptide H416–39, which had a tolerogenic effect not only on autoimmune Th cells, but autoimmune B cells as well, because this peptide contained both T and B cell autoepitopes. These studies show that the pathogenic T and B cells of lupus, despite intrinsic defects in activation thresholds, are still susceptible to autoantigen-specific tolerogens.
The SH2-Containing Inositol-5′-Phosphatase Enhances LFA-1-Mediated Cell Adhesion and Defines Two Signaling Pathways for LFA-1 Activation
Authors: Rey-Ladino Jose A. , Huber Michael , Liu Ling , Damen Jacqueline E. , Krystal Gerald , Takei Fumio
Published: January 2023

The inside-out signaling involved in the activation of LFA-1-mediated cell adhesion is still poorly understood. Here we examined the role of the SH2-containing inositol phosphatase (SHIP), a major negative regulator of intracellular signaling, in this process. Wild-type SHIP and a phosphatase-deficient mutant SHIP were overexpressed in the murine myeloid cell line, DA-ER, and the effects on LFA-1-mediated cell adhesion to ICAM-1 (CD54) were tested. Overexpression of wild-type SHIP significantly enhanced cell adhesion to immobilized ICAM-1, and PMA, IL-3, or erythropoietin further augmented this adhesion. In contrast, phosphatase dead SHIP had no enhancing effects. Furthermore, PMA-induced activation of LFA-1 on DA-ER cells overexpressing wild-type SHIP was dependent on protein kinase C but independent of mitogen-activated protein kinase activation, whereas cytokine-induced activation was independent of protein kinase C and mitogen-activated protein kinase activation but required phosphatidylinositol-3 kinase activation. These results suggest that SHIP may regulate two distinct inside-out signaling pathways and that the phosphatase activity of SHIP is essential for both of them.
Leukocyte-Associated Ig-Like Receptor-1 Functions as an Inhibitory Receptor on Cytotoxic T Cells
Authors: Meyaard Linde , Hurenkamp Jolanda , Clevers Hans , Lanier Lewis L. , Phillips Joseph H.
Published: January 2023

Leukocyte associated Ig-like receptor-1 (LAIR-1) is a surface molecule expressed on human mononuclear leukocytes that functions as an inhibitory receptor on human NK cells. In addition to NK cells, LAIR-1 is expressed on T cells, B cells, macrophages, and dendritic cells. Most cells express two biochemically distinct forms of LAIR-1, which we now show are likely alternative splice variants of the same gene. Cross-linking of LAIR-1 on human T cell clones results in inhibition of cytotoxicity only in T cell clones that lack CD28 and are able to spontaneously lyse certain targets in vitro. Moreover, the cytolytic activity of freshly isolated T cells, which is thought to be mainly due to “effector” T cells, can be inhibited by anti-LAIR-1 mAb. Thus, LAIR-1 functions as an inhibitory receptor not only on NK cells, but also on human T cells. This indicates that LAIR-1 provides a mechanism of regulation of effector T cells and may play a role in the inhibition of unwanted bystander responses mediated by Ag-specific T cells.
Functional and Phenotypic Analysis of Thymic CD34+CD1a− Progenitor-Derived Dendritic Cells: Predominance of CD1a+ Differentiation Pathway
Authors: Dalloul Ali H. , Patry Claire , Salamero Jean , Canque Bruno , Grassi Fernanda , Schmitt Christian
Published: January 2023

Whether thymic dendritic cells (DC) are phenotypically and functionally distinct from the monocyte lineage DC is an important question. Human thymic progenitors differentiate into T, NK, and DC. The latter induce clonal deletion of autoreactive thymocytes and therefore might be different from their monocyte-derived counterparts. The cytokines needed for the differentiation of DC from thymic progenitors were also questioned, particularly the need for GM-CSF. We show that various cytokine combinations with or without GM-CSF generated DC from CD34+CD1a− but not from CD34+CD1a+ thymocytes. CD34+ thymic cells generated far fewer DC than their counterparts from the cord blood. The requirement for IL-7 was strict whereas GM-CSF was dispensable but nonetheless improved the yield of DC. CD14+ monocytic intermediates were not detected in these cultures unless macrophage-CSF (M-CSF) was added. Cultures in M-CSF generated CD14−CD1a+ DC precursors but also CD14+CD1a− cells. When sorted and recultured in GM-CSF, CD14+ cells down-regulated CD14 and up-regulated CD1a. TNF-α accelerated the differentiation of progenitors into DC and augmented MHC class II transport to the membrane, resulting in improved capacity to induce MLR. The trafficking of MHC class II molecules was studied by metabolic labeling and immunoprecipitation. MHC class II molecules were transported to the membrane in association with invariant chain isoforms in CD14+ (monocyte)-derived and in CD1a+ thymic-derived DC but not in monocytes. Thus, thymic progenitors can differentiate into DC along a preferential CD1a+ pathway but have conserved a CD14+ maturation capacity under M-CSF. Finally, CD1a+-derived thymic DC and monocyte-derived DC share very close Ag-processing machinery.
The NKR-P1B Gene Product Is an Inhibitory Receptor on SJL/J NK Cells
Authors: Kung Sam K. P. , Su Ruey-Chyi , Shannon John , Miller Richard G.
Published: January 2023

The mouse NKR-P1 family includes at least three genes: NKR-P1A, -B, -C. Neither surface expression nor function of the NKR-P1B gene product has previously been shown. Here, we demonstrate that the SJL/J allele of the NKR-P1B gene product is expressed on SJL/J NK cells, and is recognized by PK136 mAb. Interestingly, the same mAb does not recognize the NKR-P1B gene product of C57BL/6. We have also generated a novel mAb, 1C10, that recognizes an activation receptor on SJL/J NK cells. Activation of the NKR-P1B receptor-inhibited 1C10 mAb induced redirected lysis and recruited SHP-1, indicating that NKR-P1B is an inhibitory receptor. Therefore, the mouse NKR-P1 gene family, like the Ly49 family, includes both activation and inhibitory receptors.