bluuebunny/crossref_metadata_2025 huggingface dataset:
CpG DNA Induces Sustained IL-12 Expression In Vivo and Resistance toListeria monocytogenesChallenge
Authors: Krieg Arthur M. , Love-Homan Laurie , Yi Ae-Kyung , Harty John T.
Published: December 2022

Vertebrates have evolved innate immune defense mechanisms that recognize and respond to structural patterns that are specific to microbial molecules. One such pattern recognition system is based on unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs); these motifs are common in bacterial DNA but are under-represented (“CpG suppression”) and methylated in vertebrate DNA. Mice that are injected with bacterial DNA or synthetic oligodeoxynucleotides (ODNs) containing CpG motifs respond with a rapid production of IL-12 and IFN-γ. The serum levels of IL-12 were increased for at least 8 days after a single injection of CpG ODNs, but IFN-γ levels returned to baseline within 24 h. This Th1-like cytokine response to CpG motifs induces a state of resistance to infection by Listeria monocytogenes in susceptible specific pathogen-free BALB/c mice. Resistance developed within 48 h of pretreatment with CpG ODNs, persisted for at least 2 wk, and was dependent upon IFN-γ secretion. These data support the hypothesis that CpG DNA motifs are a “danger signal” that activates protective innate immune defenses and may have therapeutic potential.
Novel Regulation of Cyclooxygenase-2 Expression and Prostaglandin E2 Production by IFN-γ in Human Macrophages
Authors: Barrios-Rodiles Miriam , Chadee Kris
Published: December 2022

Cyclooxygenase-2 (COX-2) is the inducible enzyme in macrophages responsible for high output PG production during inflammation and immune responses. Although several stimuli are known to regulate COX-2, the molecular mechanisms modulating its expression by the cytokine network are poorly understood. As IFN-γ priming is essential for macrophage accessory and effector cell functions, we investigated the effect of IFN-γ on COX-2 expression in U937 human macrophages stimulated with IL-1β. A dose- and time-dependent increase in COX-2 mRNA and protein expression was evoked by IL-1β, whereas the levels of COX-1, the constitutively expressed isoform, remained unaltered. Interestingly, IFN-γ-primed cells showed 40 to 60% lower levels of COX-2 mRNA, protein expression, and PGE2 production as compared with nonprimed cells. IFN-γ-priming (50–500 U/ml) down-regulated COX-2 expression in a time- and dose-dependent fashion. Furthermore, IFN-γ inhibited COX-2 gene transcription in response to IL-1β but not to LPS. In contrast, the rate of decay of COX-2 transcripts in nonprimed and primed macrophages was similar (t1/2 = 3.2 h). The down-regulatory effect of IFN-γ on IL-1β-induced COX-2 expression was abrogated with cycloheximide. These results highlight a novel mechanism of COX-2 regulation by IFN-γ at the transcriptional level, which may affect the outcome of inflammatory and immune conditions.
Bradykinin Stimulates IL-8 Production in Cultured Human Airway Smooth Muscle Cells: Role of Cyclooxygenase Products
Authors: Pang Linhua , Knox Alan J.
Published: December 2022

IL-8 is an important neutrophil and eosinophil chemoattractant in asthma. A recent report has suggested that bradykinin (BK), an asthmatic mediator, induces the release of IL-8 in nonairway cells. We have recently reported that BK causes cyclooxygenase (COX)-2 induction and PGE2 release in human airway smooth muscle (ASM) cells. In this study, we tested the ability of BK to induce IL-8 from these cells and explored the role of COX products and COX-2 induction in this process. Confluent serum-deprived human ASM cells were studied. IL-8 was assayed by specific ELISA. Unstimulated cells released low levels of IL-8. BK enhanced IL-8 release in a concentration- and time-dependent fashion (maximum 50-fold increase over basal). The nonselective COX inhibitor indomethacin and the selective COX-2 inhibitor NS-398 strongly inhibited BK-stimulated PGE2 and IL-8 production. The COX substrate arachidonic acid also caused PGE2 and IL-8 production, and its effect was inhibited by nonselective COX inhibitors but unaffected by NS-398. Both the BK- and arachidonic acid-induced IL-8 production was inhibited by the protein synthesis inhibitors cycloheximide and actinomycin D and by the steroid dexamethasone. Furthermore, exogenous PGE2 and calcium ionophore A23187 also stimulated IL-8 release. BK-induced IL-8 release was mimicked by the BK B2 receptor agonist (Tyr(Me)8)-BK and was potently inhibited by the selective B2 receptor antagonist HOE-140. These results suggest that human ASM can be a source of IL-8 and also that endogenous prostanoids, involving both COX-1 and COX-2, have a novel role in mediating BK-induced IL-8 production.
Contribution of Endothelial Selectins and α4 Integrins to Eosinophil Trafficking in Allergic and Nonallergic Inflammatory Reactions in Skin
Authors: Teixeira Mauro M. , Hellewell Paul G.
Published: December 2022

The role of endothelial selectins in mediating eosinophil recruitment was assessed using the trafficking of 111In-labeled blood eosinophils in mouse skin. An intradermal injection of chemoattractants (leukotriene B4, macrophage inflammatory protein-1α, and eotaxin) resulted in a rapid accumulation of 111In eosinophils that was reduced 49 to 91% by anti-P-selectin mAb. An anti-E-selectin mAb was ineffective, although a combined E- and P-selectin blockade resulted in >95% inhibition of all responses. The accumulation of a pulse of 111In eosinophils at sites of active cutaneous anaphylaxis (ACA) at 4 to 8 h and at 20 to 24 h after Ag challenge was completely dependent upon E- and P-selectin in combination, but not in isolation. In contrast, at 20 to 24 h after Ag challenge in a delayed-type hypersensitivity (DTH) reaction in skin, 111In eosinophil accumulation was largely independent of endothelial selectins, even when L-selectin was also blocked. An anti-α4 integrin mAb significantly reduced 111In eosinophil trafficking in both allergic reactions but was slightly more effective in the DTH reaction compared with the ACA reaction. These results show that P-selectin and to a lesser extent E-selectin mediate eosinophil recruitment in skin in acute inflammatory reactions. In allergic, late-onset inflammatory reactions, neither P- nor E-selectin alone are sufficient to mediate eosinophil accumulation; when combined, they are essential for trafficking in ACA but are less important in the DTH reaction. Whether α4 integrin-based strategies will be more effective than selectin-based strategies at inhibiting eosinophil recruitment in human disease remains to be determined.
Intracellular Expression of FcγRIII (CD16) and Its Mobilization by Chemoattractants in Human Eosinophils
Authors: Zhu Xiangdong , Hamann Kimm J. , Muñoz Nilda M. , Rubio Noel , Mayer Diane , Hernrreiter Anja , Leff Alan R.
Published: December 2022

We characterized the existence, translocation, and reabsorption during cellular activation of a constitutively expressed intracellular CD16 in the human eosinophil. By two-color flow cytometry, we showed that 6.5 ± 0.3% of nonpurified eosinophils expressed surface CD16. After digestion with phosphotidylinositol-specific phospholipase C, surface CD16 on both neutrophils and eosinophils decreased substantially, suggesting that eosinophil CD16 is a glycosyl-phosphatidylinositol-linked isoform. However, CD16 was substantially expressed intracellularly in human eosinophils. Epitope-specific binding to CLB-gran11 mAb from non-NA2/NA2 donors demonstrated that intracellular eosinophil CD16 also differed from the transmembrane isoform of CD16 expressed on NK cells or macrophages. Western blot analysis performed with 3G8 or DJ130c mAb showed a broad band at ∼65 to 80 kDa, which was the same as neutrophil CD16 from the same NA2/NA2 donors. Upon stimulation by chemoattractants C5a, FMLP, or platelet-activating-factor, eosinophilic intracellular CD16 was rapidly translocated to the eosinophil surface, expressed maximally at 30 s, and then gradually disappeared from the cell surface during the next 10 min. Intracellular flow cytometry of stimulated eosinophils and sandwich ELISA of stimulated eosinophil supernatants demonstrated that the disappearance was due to its rapid release into medium and reabsorption by the cells. Our data identify a CD16B that is consistently expressed intracellularly but only rarely on the surface of nonactivated human eosinophils. This CD16 is transiently expressed during stimulation by chemoattractants.
Histamine Potently Suppresses Human IL-12 and Stimulates IL-10 Production via H2 Receptors
Authors: Elenkov Ilia J. , Webster Elizabeth , Papanicolaou Dimitris A. , Fleisher Thomas A. , Chrousos George P. , Wilder Ronald L.
Published: December 2022

IL-12 and IL-10, respectively, stimulate Th1 and Th2 immune responses. The development of some allergic reactions, infections, and tumors are associated with excessive histamine production and a shift toward Th2 responses. Here we address the possibility that this association is causally linked, at least in part, to modulation of IL-12 and IL-10 production by histamine. We report that histamine dose-dependently inhibited the secretion of human IL-12 (p70) and increased the production of IL-10 in LPS-stimulated whole blood cultures. These effects of histamine were antagonized by cimetidine, an H2 receptor antagonist, but not by selective H1 and H3 receptor blockers, and were mimicked by an H2 receptor agonist. The effects of histamine on IL-12 and IL-10 secretion were independent of endogenous secretion of IL-10 or exogenous addition of IL-12, while Ro 20–1724, a phosphodiesterase inhibitor, potentiated the effects of histamine on IL-12 and IL-10 production, implicating cAMP in its actions. Similar modulatory effects of histamine on IL-12 and IL-10 production, which were reversed by the H2 antagonist cimetidine, were observed in PBMC and isolated monocytes stimulated by Staphylococcus aureus Cowan strain 1 and LPS, respectively. Thus, histamine, via stimulation of H2 receptors on peripheral monocytes and subsequent elevation of cAMP, suppresses IL-12 and stimulates IL-10 secretion, changes that may result in a shift of Th1/Th2 balance toward Th2-dominance. This may represent a novel mechanism by which excessive secretion of histamine potentiates Th2-mediated allergic reactions and contributes to the development of certain infections and tumors normally eliminated by Th1-dependent immune mechanisms.
Impaired Yield, Phenotype, and Function of Monocyte-Derived Dendritic Cells in Humans at Risk for Insulin-Dependent Diabetes
Authors: Takahashi Kazuma , Honeyman Margo C. , Harrison Leonard C.
Published: December 2022

Dendritic cells (DC) present Ag to naive T cells and are therefore pivotal in shaping immune responses. DC may either immunize or tolerize T cells. Humans with pancreatic islet autoimmunity at high risk for insulin-dependent diabetes mellitus (IDDM) present the opportunity to investigate DC in autoimmune disease. We compared DC phenotype and function in 12 euglycemic, asymptomatic IDDM relatives with islet autoimmunity and controls matched for age, sex, and MHC class II alleles. DC were generated from adherent peripheral blood cells by culture with granulocyte/macrophage-CSF and IL-4. The yield of DC was significantly lower in IDDM relatives than in controls. While the DC phenotype, HLA-DR+CD14−, was expressed by ≥90% of the cells generated from relatives and controls, the proportion of cells that expressed CD1a and the costimulator molecules CD80 (B7-1) and CD86 (B7-2) was significantly lower in IDDM relatives. In addition, B7-1 and B7-2 expression per cell was significantly lower in IDDM relatives. These phenotypic changes were accompanied by reduced stimulation of autologous CD4 cells by DC from IDDM relatives. Similar findings were obtained in three recently diagnosed IDDM patients. These findings indicate that impairment of DC phenotype and function is a marker of islet autoimmunity and are consistent with a role for impaired DC function in the pathogenesis of autoimmune disease.
IA-2 (Islet Cell Antigen 512) Is the Primary Target of Humoral Autoimmunity Against Type 1 Diabetes-Associated Tyrosine Phosphatase Autoantigens
Authors: Bonifacio Ezio , Lampasona Vito , Bingley Polly J.
Published: December 2022

IA-2 (islet cell Ag 512) and IA-2β (phogrin/IAR) are related autoantigens associated with type 1 diabetes. To determine the critical regions for autoantibody binding and which of these autoantigens is the primary target, mutant and chimeric constructs were used to characterize Ab epitope binding in sera from 217 new onset patients with type 1 diabetes and sequential samples from 141 islet cell Ab positive first degree relatives of patients. All 22 relatives and 121 of 129 patients with IA-2/IA-2β Abs had reactivity to IA-2-specific epitopes. These epitopes were in the juxtamembrane region (residues 601–682) and the protein tyrosine phosphatase (PTP)-like domain of IA-2. Chimeras showed that IA-2 residues 795–889 were important for IA-2-specific Ab binding in the PTP-like domain, and mutation of IA-2 residues 877 and 911, previously indicated as relevant for phosphatase activity, also reduced Ab binding. In contrast, Ab binding to IA-2β was limited to its PTP-like domain, most IA-2β Abs recognized epitopes shared with IA-2, and only 20 patients and 2 relatives had Abs to IA-2β-specific epitopes. In 4 relatives, IA-2 and/or IA-2β Abs developed in follow-up samples. In each of these, Abs to IA-2-specific epitopes were the first detected. In three, spreading to epitopes shared between IA-2 and IA-2β in subsequent samples was seen. In the 17 relatives who developed type 1 diabetes, progression to disease was associated with reactivity to multiple IA-2/IA-2β epitopes. These data suggest that IA-2 is the primary phosphatase-like autoantigen associated with type 1 diabetes and that studying autoantibody epitope diversity may assist in disease prediction.
Cutting Edge Commentary: Chemokine Regulation of Experimental Autoimmune Encephalomyelitis: Temporal and Spatial Expression Patterns Govern Disease Pathogenesis
Authors: Karpus William J. , Ransohoff Richard M.
Published: December 2022

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ Th1-mediated demyelinating disease of the central nervous system that serves as a model for multiple sclerosis (MS). There are several considerations that suggest a role for chemokines in the disease process. First, chemokines are highly expressed in the central nervous system with a tight temporal relationship to disease activity. Second, in vivo neutralization studies showed a distinct role for specific chemokines in the evolution of the process. Third, the selective and differential expression of chemokines in differing models of EAE bears a close relationship to the patterns of inflammatory pathology. Fourth, the spatial distribution of chemokine expression could plausibly contribute to lesion architecture. Finally, preliminary observations in MS material suggest that chemokine expression observed in EAE may provide useful information regarding the pathogenesis of inflammation in MS. We propose that temporal and spatial expression of chemokines are crucial factors, complementing adhesion molecule up-regulation, that regulate EAE disease activity.
HindIII Liposomes Suppress Delayed-Type Hypersensitivity Responses In Vivo and Induce Epidermal IL-10 In Vitro
Authors: Nishigori Chikako , Yarosh Daniel , O’Connor Adrienne , K. Shreedhar Vijay , Ullrich Stephen E. , Cox Patricia , Kripke Margaret L.
Published: December 2022

Considerable evidence suggests that ultraviolet-B (UV-B) radiation suppresses certain immune responses through the induction of cyclobutane pyrimidine dimers in DNA. To determine whether induction of other forms of DNA damage in the skin mimicked the immunosuppressive effects of UV-B radiation, we produced double-strand breaks in the DNA of epidermal cells with HindIII restriction endonuclease encapsulated in liposomes. Application of these liposomes, but not liposomes containing inactive HindIII or an irrelevant endonuclease, to the skin of C3H mice suppressed the induction of delayed-type hypersensitivity responses to Candida albicans and alloantigen and induced IL-10 production in the epidermis. Treatment of the Pam212 murine keratinocyte cell line with these liposomes in vitro induced immunosuppressive activity and IL-10 in culture supernatants. Unlike UV-B irradiation, however, HindIII in liposomes failed to induce suppressor T cell activity in vivo or in vitro. We conclude that double-strand breaks in DNA of epidermal cells can induce immunosuppression and up-regulate the production of immunomodulatory cytokines; however, either DNA damage alone does not account for all the immunosuppressive properties of UV-B irradiation, or cyclobutane pyrimidine dimers differ qualitatively from double-strand breaks in their biologic consequences. These studies raise the possibility that drugs causing DNA damage may induce cytokine dysregulation and immune suppression in addition to cytotoxicity.
B Cell Tolerance to a Minor, But Not to a Major, Antigenic Surface of the Self Antigen, Cytochromec
Authors: Jemmerson Ronald , Minnerath Jeanne M. , Hedrick Stephen M. , Oehen Stephan
Published: December 2022

To study B cell tolerance to the mitochondrial protein cytochrome c (CYT), the B cell response to pigeon CYT (PCC) was examined in mice transgenic for PCC. PCC was coupled to OVA to provide T cell help, since PCC-specific T cells in PCC-transgenic mice are deleted in the thymus. The frequency of secondary B cells responding to the minor antigenic surface around residue 44 on PCC was decreased about 10-fold in native PCC-transgenic mice compared with that in control mice or in transgenic mice expressing an altered form of PCC that lacked the heme and had a different amino acid sequence at the N-terminus. A similar decrease has been observed in the frequency of B cells in normal mice recognizing the site around residue 44 on mouse CYT compared with the frequency of B cells recognizing the corresponding site on foreign CYT. There were no major decreases but apparently were compensatory increases in the frequencies of B cells recognizing other sites on PCC in the native PCC-transgenic mice compared with those in other mice. These results indicate that B cells in mice are only partially tolerant to self CYT. A possible basis for this partial tolerance relating to the fate of CYT in cell death is discussed. This may be the first example of the use of a transgenic system to study B cell tolerance to a homologous self Ag.
IL-13 Suppresses TNF-Induced Activation of Nuclear Factor-κB, Activation Protein-1, and Apoptosis
Authors: Manna Sunil K. , Aggarwal Bharat B.
Published: December 2022

IL-13 is known to suppress the production of inflammatory cytokines such as TNF. Whether IL-13 also modulates the biologic effects of TNF is not known. In the present report we examined the effect of IL-13 on TNF-induced activation of nuclear transcription factors NF-κB and activation protein-1 (AP-1) and apoptosis. Pretreatment of cells with IL-13 blocked TNF-induced NF-κB activation, nuclear translocation of p65 subunit, and degradation of IκBα. IL-13 also inhibited NF-κB activation by LPS, okadaic acid, H2O2, and ceramide. TNF-induced NF-κB-dependent gene transcription was also blocked by IL-13. TNF-induced activation of another nuclear transcription factor, AP-1, was suppressed by IL-13. The activation of N-terminal c-Jun kinase and mitogen-activated protein kinase kinase, implicated in the regulation of AP-1 and NF-κB, was also down-regulated by IL-13. TNF-mediated cytotoxicity and activation of caspase-3 were abolished by IL-13. The inhibitory effects of IL-13 on TNF were sensitive to H-7, neomycin, and wortmannin, suggesting that the pathway consisting of protein kinase C, phosphatidylinositol 3-kinase, and phospholipase C must be involved in IL-13 signaling. Thus, overall, these results demonstrate that IL-13 is a potent inhibitor of TNF-mediated activation of NF-κB, AP-1, and apoptosis, which may contribute to its previously described immunosuppressive and anti-inflammatory effects.
HLA-DR4 (DRB1*0401) Transgenic Mice Expressing an Altered CD4-Binding Site: Specificity and Magnitude of DR4-Restricted T Cell Response
Authors: Pan Shuchong , Trejo Theodore , Hansen Julie , Smart Michele , David Chella S.
Published: December 2022

Optimum function of HLA-DR molecules in transgenic mice requires efficient interaction between the class II molecules on APCs and CD4 on T cells. Residues 110 and 139 of the second domain of class II molecules are considered to be critical for recognition of CD4. We generated an HLA-DR4β(NT) transgene construct in which positions 110 and 139 were altered to resemble endogenous mouse H2 Aβ molecules. This construct was introduced into (B10 × SWR) embryos, and DR4β(NT) transgenic mice were produced. The transgene was transferred into B10.RFB3 (Eβ0 Eαp) mice. The transgene-encoded DR4β molecules paired with endogenous Eα chains to form stable DR4β/Eα dimers expressed on the cell surface. The hybrid dimers showed similar Ag-binding specificity to HLA-DR4 molecules and positively selected CD4+ T cells in vivo. Immunization of HLA-DR4β(NT) transgenic mice with DR4-restricted peptides induced T cell proliferation in vitro. While the purified T cells from DR4β(NT) transgenic mice responded strongly to the HA(307–319) presented by M12C3 transfectants expressing altered DR4β/Eα heterodimers, the response to the same peptides presented by transfectants expressing wild-type DR4β/Eα molecules was substantially reduced. Taken together, these data confirmed in vitro studies on the importance of these residues in CD4-MHC class II interaction. The altered HLA-DR4β transgenic mice were able to overcome the species barrier and generate efficient HLA-DR4-restricted CD4-specific immune responses. Thus, residues 110 and 139 were critical for the interaction of class II with CD4 T cells during thymic selection as well as peripheral immune responses.
Limited Diversity of Human scFv Fragments Isolated by Panning a Synthetic Phage-Display scFv Library with Cultured Human Melanoma Cells
Authors: Noronha Elvyra J. , Wang Xinhui , Desai Smruti A. , Kageshita Toshiro , Ferrone Soldano
Published: December 2022

To broaden the specificity of the Abs recognizing human melanoma-associated Ags (MAAs), we have isolated human single-chain fragment of the V region (scFv) fragments by panning the synthetic phage Ab library (#1) with the human melanoma cell lines S5 and SK-MEL-28. All of the isolated scFv fragments reacted with the mouse mAb defined high molecular weight melanoma-associated Ag (HMW-MAA). scFv #70 immunoprecipitates the two characteristic subunits of HMW-MAA, while scFv #28 only immunoprecipitates its large subunit. These results challenge the current view regarding the structure of HMW-MAA and indicate that it consists of two independent subunits. The human scFv fragments share some similarities with the mouse anti-HMW-MAA mAb. Like mAb 149.53 and 225.28, scFv #28 reacts with rat B49 neural cells that express a homologue of HMW-MAA. scFv #70 reacts with a determinant that is spatially close to the one identified by mAbs 149.53, VT68.2, and VT86. Besides suggesting similarities in the recognition of human melanoma cells by the mouse and human Ab repertoire, these results indicate that the Abs isolated from synthetic Ab libraries resemble those that are found in natural Ab repertoires. The restricted diversity of the scFv fragments that were isolated by panning synthetic Ab libraries with different melanoma cell lines suggests that certain Ags, like HMW-MAA, are immunodominant in vitro. This phenomenon, which parallels the in vivo immunodominance of certain Ags, implies that the antigenic profile of the cells used for panning determines the specificity of the preponderant population of isolated Abs.
Inducible Nitric Oxide Synthase Is Not Required for Long-Term Vaccine-Based Immunity AgainstToxoplasma gondii
Authors: Khan Imtiaz A. , Matsuura Tadashi , Kasper Lloyd H.
Published: December 2022

Induction of reactive nitrogen intermediates by IFN-γ is presumed an important mechanism of host resistance against acute and chronic infection with Toxoplasma gondii. Although nitric oxide (NO) has been shown to be important in the control of parasite replication in vivo, the role of this molecule in vaccine-based immunity against T. gondii is unknown. Mice with a targeted disruption of inducible NO synthase (iNOS) were immunized with an avirulent temperature-sensitive strain of this parasite (ts-4). Both the parental C57BL/6 and the iNOS−/− mice survived infection with the ts-4 mutant. Oral challenge of the vaccinated mice with a lethal dose of cysts containing bradyzoites resulted in reduced parasite burden and increased survival compared with nonvaccinated control mice. Host immunity in the iNOS−/− mice, similar to that observed in the parental strain, appears dependent upon both IFN-γ and CD8+ T cells. These findings suggest that although vaccine-based long-term immunity against T. gondii is dependent upon the induction of IFN-γ, it does not rely upon the anti-microbial effect of NO.
Prostaglandin E2 Protects Against Liver Injury AfterEscherichia coliInfection but Hampers the Resolution of the Infection in Mice
Authors: Takano Manabu , Nishimura Hitoshi , Kimura Yuki , Washizu Junji , Mokuno Yasujii , Nimura Yuji , Yoshikai Yasunobu
Published: December 2022

cAMP-increasing agents such as prostaglandin E2 (PGE2) are known to protect against LPS-induced liver injury by down-regulating the production of inflammatory cytokines such as TNF-α. However, the effects of such reagents on host defense against bacterial infection remain unknown. We show here that in vivo administration of PGE2 significantly protected mice against liver injury after Escherichia coli infection but hampered the resolution of the infection. PGE2 significantly suppressed circulating TNF-α and IL-12 levels but increased the IL-10 production after E. coli challenge. PGE2 inhibited the emergence of γδ T cells in the peritoneal cavity, which are important for host defense against E. coli, and deteriorated bacterial exclusion in the peritoneal cavity after E. coli challenge. These results suggested that PGE2 affects host defense mechanisms against E. coli infection through modulation of cytokine production and γδ T cell accumulation.
Murine IgG1 Complexes Trigger Immune Effector Functions Predominantly via FcγRIII (CD16)
Authors: Hazenbos Wouter L. W. , Heijnen Ingmar A. F. M. , Meyer Dirk , Hofhuis Frans M. A. , Renardel de Lavalette Chantal , Schmidt Reinhold E. , Capel Peter J. A. , van de Winkel Jan G. J. , Gessner J. Engelbert , van den Berg Timo K. , Verbeek J. Sjef
Published: December 2022

Previously, we have demonstrated that phagocytosis of IgG1-coated particles by macrophages in vitro is impaired by deletion of FcγRIII in mice, suggesting that IgG1 may interact preferentially with FcγRIII. In the present study, the biologic relevance of this observation was addressed by triggering various effector functions of the immune system in FcγRIII−/− mice, using panels of mAbs of different IgG subclasses. Both binding and phagocytosis of IgG1-coated sheep or human erythrocytes by FcγRIII−/− macrophages in vitro were strongly impaired, indicating that the impaired ingestion of complexed IgG1 by FcγRIII−/−macrophages is due to a defect in binding. An in vivo consequence of the defective phagocytosis was observed by resistance of FcγRIII-deficient mice to experimental autoimmune hemolytic anemia, as shown by a lack of IgG1-mediated erythrophagocytosis in vivo by liver macrophages. Furthermore, trapping of soluble IgG1-containing immune complexes by follicular dendritic cells in mesenteric lymph nodes from FcγRIII−/− mice was abolished. Whole blood from FcγRIII−/− mice was unable to induce lysis of tumor cells in the presence of IgG1 antitumor Abs. Finally, IgG1 mAbs proved unable to mount a passive cutaneous anaphylaxis in FcγRIII−/− mice. Together, these results demonstrate that IgG1 complexes, either in particulate or in soluble form, trigger in vitro and in vivo immune effector functions in mice predominantly via FcγRIII.
CXCR4 and CCR5 on Human Thymocytes: Biological Function and Role in HIV-1 Infection
Authors: Zaitseva Marina B. , Lee Shirley , Rabin Ronald L. , Tiffany H. Lee , Farber Joshua M. , Peden Keith W. C. , Murphy Philip M. , Golding Hana
Published: December 2022

Thymocyte infection with HIV-1 is associated with thymic involution and impaired thymopoiesis, particularly in pediatric patients. To define mechanisms of thymocyte infection, we examined human thymocytes for expression and function of CXCR4 and CCR5, the major cell entry coreceptors for T cell line-tropic (T-tropic) and macrophage-tropic (M-tropic) strains of HIV-1, respectively. CXCR4 was detected on the surface of all thymocytes. CXCR4 expression on mature, high level TCR thymocytes was similar to that on peripheral blood T cells, but was much lower than that on immature thymocytes, including CD34+ thymic progenitors. Consistent with this, stroma-derived factor-1 (SDF-1) induced calcium flux primarily in immature thymocytes, with CD34+ progenitors giving the strongest response. In addition, SDF-1 mRNA was detected in thymic-derived stromal cells, and SDF-1 induced chemotaxis of thymocytes, suggesting that CXCR4 may play a role in thymocyte migration. Infection of immature thymocytes by the T-tropic HIV-1 strain LAI was 10-fold more efficient than that in mature thymocytes, consistent with their relative CXCR4 surface expression. Anti-CXCR4 antiserum or SDF-1 blocked fusion of thymocytes with cells expressing the LAI envelope. In contrast to CXCR4, CCR5 was detected at low levels on thymocytes, and CCR5 agonists did not induce calcium flux or chemotaxis in thymocytes. However, CD4+ mature thymocytes were productively infected with the CCR5-tropic strain Ba-L, and this infection was specifically inhibited with the CCR5 agonist, macrophage inflammatory protein-1β. Our data provide strong evidence that CXCR4 and CCR5 function as coreceptors for HIV-1 infection of human thymocytes.
Signaling Through a CD3γ-Deficient TCR/CD3 Complex in Immortalized Mature CD4+ and CD8+ T Lymphocytes
Authors: Pacheco-Castro Alberto , Alvarez-Zapata David , Serrano-Torres Pilar , Regueiro José R.
Published: December 2022

The biologic role of each CD3 chain and their relative contribution to the signals transduced through the TCR/CD3 complex and to downstream activation events are still controversial: they may be specialized or redundant. We have immortalized peripheral blood CD4+ and CD8+ T lymphocytes from a human selective CD3γ deficiency using Herpesvirus saimiri. The accessibility of the mutant TCR/CD3 complex to different Abs was consistently lower in immortalized CD8+ cells when compared with CD4+ cells, relative to their corresponding CD3γ-sufficient controls. Several TCR/CD3-induced downstream activation events, immediate (calcium flux), early (cytotoxicity and induction of surface CD69 or CD40L activation markers or intracellular TNF-α) and late (proliferation and secretion of TNF-α), were normal in γ-deficient cells, despite the fact that their TCR/CD3 complexes were significantly less accessible than those of controls. In contrast, the accumulation of intracellular IL-2 or its secretion after CD3 triggering was severely impaired in γ-deficient cells. The defect was upstream of protein kinase C activation because addition of transmembrane stimuli (PMA plus calcium ionophore) completely restored IL-2 secretion in γ-deficient cells. These results suggest that the propagation of signals initiated at the TCR itself can result in a modified downstream signaling cascade with distinct functional consequences when γ is absent. They also provide evidence for the specific participation of the CD3γ chain in the induction of certain cytokine genes in both CD4+ and CD8+ human mature T cells. These immortalized mutant cells may prove to be useful in isolating cytosolic signaling pathways emanating from the TCR/CD3 complex.
SLP-76 Expression Is Restricted to Hemopoietic Cells of Monocyte, Granulocyte, and T Lymphocyte Lineage and Is Regulated During T Cell Maturation and Activation
Authors: Clements James L. , Ross-Barta Susan E. , Tygrett Lorraine T. , Waldschmidt Thomas J. , Koretzky Gary A.
Published: December 2022

The leukocyte-specific adapter protein SLP-76 is known to augment the transcriptional activity of nuclear factor of activated T cells and AP-1 following TCR ligation. A role for SLP-76 in additional receptor-mediated signaling events is less clear. To define the pattern of SLP-76 expression during murine hemopoiesis, we stained cells isolated from various tissues with a combination of surface markers followed by intracellular staining with a fluorochrome-labeled SLP-76-specific Ab. In the bone marrow, SLP-76 expression is largely restricted to cells of granulocyte and monocyte lineage. Heterogeneous SLP-76 expression is first detected in the CD44+CD25− subset within the CD3−CD4−CD8− thymocyte population. Interestingly, SLP-76 expression increases as thymocyte maturation progresses within the CD4−CD8− compartment but decreases as cells mature to a CD4+CD8+ phenotype. SLP-76 expression is then up-regulated following selection and concomitant with maturation to a CD4+ or CD8+ phenotype. In the periphery, SLP-76 is expressed in T lymphocytes with no detectable expression in the B cell compartment. Exposure to the superantigen staphylococcal enterotoxin B augments SLP-76 expression in the reactive T cell subset. Furthermore, in vitro stimulation with TCR-specific Abs augments the existing levels of SLP-76. These data reveal that SLP-76 expression is coordinately regulated with surface expression of a pre-TCR or mature TCR complex during thymocyte development and that TCR ligation elicits signals that result in increased expression of SLP-76.
Effects of Bacterial DNA on Cytokine Production by (NZB/NZW)F1 Mice
Authors: Gilkeson Gary S. , Conover Jacqueline , Halpern Melissa , Pisetsky David S. , Feagin Amy , Klinman Dennis M.
Published: December 2022

Microbial DNA has multiple immune effects including the capacity to induce polyclonal B cell activation and cytokine production in normal mice. We recently described the accelerated induction of anti-DNA Abs in NZB/NZW mice immunized with Escherichia coli (EC) dsDNA; paradoxically these mice developed less renal disease than unimmunized mice or mice immunized with calf thymus DNA. We postulated that alterations in cytokine production induced by bacterial DNA may play a key role in renal protection. To determine the effect of bacterial DNA on cytokine production in NZB/NZW mice, we measured the serum cytokine levels, cell culture supernatant cytokine levels, and number of cytokine-producing splenocytes in NZB/NZW mice injected with EC DNA, calf thymus DNA, or an immune active oligonucleotide. There was a 10- to 25-fold increase in the number of cells secreting IFN-γ compared with IL-4 in mice immunized with EC DNA. IL-12-secreting cells were also increased by bacterial DNA immunization. In parallel with the increase in IFN-γ secreting cells, there was a significant rise in serum IFN-γ levels in mice receiving EC DNA. These results indicate that EC DNA modulates systemic cytokine levels in NZB/NZW mice, selectively increasing IL-12 and IFN-γ while decreasing IL-4 production. The cytokine response of NZB/NZW mice to bacterial DNA may be of significance in disease pathogenesis and relevant to the treatment of lupus-like disease.
Development of Peptide-Selected CD8 T Cells in Fetal Thymic Organ Culture Occurs via the Conventional Pathway
Authors: Hogquist Kristin A. , Bonnevier Jody L.
Published: December 2022

Fetal thymic organ culture of TCR transgenic (Tg) tissue has been used to study issues of timing and specificity in T cell development. Because most TCR Tgs express a rearranged αβ TCR on the cell surface at an earlier stage in development than normal mice, there is a possibility that the conclusions of studies using TCR Tg cultures may not apply to normal development. In particular, in our studies of peptide-induced development of CD8 T cells, it is possible that the peptide acts on the immature double-negative cell, driving development of CD8 T cells without passing through a double-positive stage. This issue was examined by asking whether MHC class I restriction was required and by analyzing CD8β levels and endogenous TCRα chain rearrangements. We found that if nonstimulatory peptides were used in fetal thymic organ culture, CD8 T cells developed via the conventional pathway, transiting through a double-positive stage. However, we could not rule out that cells selected in the presence of stimulatory peptides (agonists) did not develop directly from double-negative precursors.
Positive and Negative Regulation of Human T Cell Activation Mediated by the CTLA-4/CD28 Ligand CD80
Authors: Boulougouris George , McLeod Julie D. , Patel Yusuf I. , Ellwood Claire N. , Walker Lucy S. K. , Sansom David M.
Published: December 2022

CD28 and CTLA-4 are related receptors that differentially regulate T cell activation. Despite the fact that they bind the same ligands, CD28 is a classical costimulator enhancing proliferation whereas CTLA-4 appears to perform negative regulatory functions. In this study, we have utilized the natural ligand for CD28 and CTLA-4 (CD80) to determine under what circumstances positive and negative effects are operative. We show here that the stimulation of purified human T cells with phorbol ester and ionomycin is inhibited in the presence of Chinese hamster ovary (CHO) cells expressing CD80. This inhibition is reversed by blocking with both anti-CD80 or Fab fragments of anti-CTLA-4 but also requires CD28 engagement. Furthermore, we show that the inhibitory function of CD80 requires elevated intracellular calcium since inhibition was observed only in the presence of ionomycin. In the absence of intracellular calcium elevation, CTLA-4 was not expressed at the cell surface, and CD80 acted positively as a costimulator of T cells, via CD28. These results demonstrate that the natural ligand CD80 can either costimulate or inhibit T cell responses depending on the conditions of T cell stimulation.
Initiation of the Autologous Mixed Lymphocyte Reaction Requires the Expression of Costimulatory Molecules B7-1 and B7-2 on Human Peripheral Blood Dendritic Cells
Authors: Scheinecker Clemens , Machold Klaus P. , Majdic Otto , Höcker Paul , Knapp Walter , Smolen Josef S.
Published: December 2022

The human autologous mixed lymphocyte reaction (AMLR) consists of a proliferative response of primarily CD4+ T lymphocytes stimulated by autologous non-T cells expressing class II MHC-encoded gene products and is thought to represent a self-recognitive mechanism that might be important in regulating the cellular interactions involved in the generation of normal immune responses. To further define appropriate stimulator cell populations, as well as the molecular mechanism responsible for the initiation of AMLR, we compared the T cell-stimulatory capacity of highly purified populations of peripheral blood dendritic cells (DCs) and monocytes (Mos) under serum-free conditions, thus carefully avoiding the presence of xenogeneic Ags. Whereas both freshly isolated Mos and DCs were found to be poor stimulators of autologous T cell proliferation, preactivation of DCs, but not of Mos, for 48 h with granulocyte-macrophage CSF led to a 113-fold increase in DC stimulatory capacity. AMLR was inhibited by mAbs against HLA-DR and CD4 molecules, and, in addition, showed a higher dependence on the granulocyte-macrophage CSF-induced up-regulation and/or de novo expression of the costimulatory molecules B7-2 and, in particular, B7-1 as compared with an Ag-specific or allogeneic MLR. Thus, our data suggest that the high density of costimulatory molecules together with MHC class II molecules on competent APCs appear to be the major triggers for the initiation of AMLR.
Deletion of Bone Marrow Stromal Cell Antigen-1 (CD157) Gene Impaired Systemic Thymus Independent-2 Antigen-Induced IgG3 and Mucosal TD Antigen-Elicited IgA Responses
Authors: Itoh Motoyuki , Ishihara Katsuhiko , Hiroi Takachika , Ok Lee Byung , Maeda Hisoka , Iijima Hideki , Yanagita Manabu , Kiyono Hiroshi , Hirano Toshio
Published: December 2022

Bone marrow stromal cell Ag-1 (BST-1; CD157)-deficient mice were generated to examine the immunologic roles of the molecule in vivo. In BST-1−/− mice, the development of peritoneal B-1 cells was delayed, and CD38low/− B-lineage cells were increased in the bone marrow and spleen. Partial impairment of thymus-independent (TI-2) and thymus-dependent (TD) Ag-specific immune responses was noted in the systemic and mucosal compartments of BST-1−/− mice, respectively. Although serum Ig levels as well as TD and TI-1 Ag-specific systemic immune responses were normal, the TI-2 Ag-induced IgG3 response was selectively impaired. Oral immunization of BST-1−/− mice with cholera toxin, a potent TD Ag for the induction of IgA response, resulted in the poor production of Ag-specific Abs at the intestinal mucosa accompanied by the reduced number of Ag-specific IgA-producing cells in the lamina propria. These results indicate that BST-1 has roles in B cell development and Ab production in vivo.
Lipid Vesicle Size Determines the Th1 or Th2 Response to Entrapped Antigen
Authors: Brewer James M. , Tetley Laurence , Richmond James , Liew Foo Y. , Alexander James
Published: December 2022

Understanding the factors that control the differential induction of Th1 and Th2 responses is a key immunologic objective with profound implications for vaccination and immunotherapy of infectious and autoimmune diseases. Using Ag formulated in lipid vesicles prepared from nonionic surfactants, we describe a novel mechanism influencing the balance of the Th1 or Th2 response. Our results indicate that inoculation of BALB/c mice with vesicles with a mean diameter ≥225 nm preferentially induces Th1 responses, as characterized by increased titers of IgG2a in plasma and elevated IFN-γ production by lymph node cells. However, preparation of the same quantity of Ag in vesicles with mean diameter of ≤155 nm induces a Th2 response, as identified by IgG1 in the absence of IgG2a production and increased lymph node IL-5 production. Although large (≥225 nm) vesicles could induce IL-12 production, smaller vesicles (≤155 nm) could not. However, small vesicles did induce higher levels of IL-1β production by macrophages than larger vesicles. The role of IL-12 in this response was confirmed in IL-12-deficient mice, whose spleen cells failed to produce IFN-γ following in vivo priming with Ag prepared in large vesicles. Our results therefore indicate that macrophages respond to endocytosis of large or small vesicles by producing different patterns of cytokines that can subsequently direct the immune response toward a Th1 or a Th2 phenotype.
The Role of Ets-1 in Mast Cell Granulocyte-Macrophage Colony-Stimulating Factor Expression and Activation
Authors: McKinlay Leigh H. , Tymms Martin J. , Thomas Ross S. , Seth Arun , Hasthorpe Suzanne , Hertzog Paul J. , Kola Ismail
Published: December 2022

Ets-1 is a transcription factor with restricted expression in lymphocytes, and it has been implicated in the regulation of T cell genes such as TCRα, TCRβ, CD4, IL-2, and TNF-α. We show in this study that Ets-1 is also expressed in some mast cells constitutively and can be induced in primary mast cells with stimuli that activate mast cells. We also show that Ets-1 plays a role in the regulation of granulocyte-macrophage CSF (GM-CSF), a cytokine expressed by activated mast cells. We have characterized a murine growth factor-independent mast cell line, FMP6−, derived from a factor-dependent cell line, FMP1.6. FMP6− has acquired a distinct connective tissue mast cell-like phenotype, as characterized by the expression of mast cell proteases MMCP-4 and MMCP-6, expression of IL-12, and the down-regulation of IL-4. The parental FMP1.6 cell line displays a mucosal mast cell-like phenotype. FMP6− cells have increased Ets-1 expression and achieve growth-factor independence by the autocrine production of GM-CSF and IL-3. Transient transfection of an Ets-1 expression construct in FMP6− cells results in transactivation of a GM-CSF reporter, while a point mutation in the consensus Ets binding site in the conserved lymphokine element, CLE0, abolishes Ets-1 transactivation. Importantly, antisense Ets-1 demonstrates an ability to repress the activity of the GM-CSF reporter. These data suggest a role for Ets-1 in mast cell growth regulation and activation, and because of the central role of mast cells in inflammatory processes, such as asthma and rheumatoid arthritis, they identify Ets-1 as potentially contributing to the pathophysiology of such diseases.
Nitric Oxide Prevents IL-1β and IFN-γ-Inducing Factor (IL-18) Release from Macrophages by Inhibiting Caspase-1 (IL-1β-Converting Enzyme)
Authors: Kim Young-Myeong , Talanian Robert V. , Li Jianrong , Billiar Timothy R.
Published: December 2022

Procytokine processing by caspase-1 is required for the maturation and release of IL-1β and IFN-γ-inducing factor (IGIF) (or IL-18) from activated macrophages (Mφ). Nitric oxide (NO) has emerged as a potent inhibitor of cysteine proteases. Here, we tested the hypothesis that NO regulates cytokine release by inhibiting IL-1β-converting enzyme (ICE) or caspase-1 activity. Activated RAW264.7 cells released four to five times more IL-1β, but not TNF-α, in the presence of the NO synthase inhibitor NG-monomethyl-l-arginine. Stimulated peritoneal Mφ from wild-type mice (inducible NO synthase (iNOS)+/+) also released more IL-1β if exposed to NG-monomethyl-l-arginine, whereas Mφ from iNOS knockout mice (iNOS−/−) did not. Inhibition of NO synthesis in stimulated RAW264.7 cells also resulted in a threefold increase in intracellular caspase-1 activity. The NO donor S-nitroso-N-acetyl-dl-penicillamine inhibited caspase-1 activity in cells as well as the activity of purified recombinant caspase-1 and also prevented the cleavage of pro-IL-1β and pro-IGIF by recombinant caspase-1. The inhibition of caspase-1 by NO was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. An in vivo role for the regulation of caspase-1 by NO was established in iNOS knockout animals, which exhibited significantly higher plasma levels of IL-1β and IFN-γ than their wild-type counterparts at 10 h following LPS injection. Taken together, these data indicate that NO suppresses IL-1β and IGIF processing by inhibiting caspase-1 activity, providing evidence for a unique role for induced NO in regulating IL-1β and IGIF release.
Down-Regulation of Tumoricidal NK and NK T Cell Activities by MHC Kb Molecules Expressed on Th2-Type γδ T and αβ T Cells Coinfiltrating in Early B16 Melanoma Lesions
Authors: Seo Naohiro , Tokura Yoshiki , Furukawa Fukumi , Takigawa Masahiro
Published: December 2022

We examined whether γδ T and αβ T cells accumulating in early B16 melanoma lesions regulate NK and NK T cells that attack tumor cells. Freshly isolated and cultured tumor-infiltrating lymphocyte (TIL) populations of NK and NK T cells lysed B16 and produced IFN-γ, whereas γδ T and a large part of αβ T cell populations had no substantial cytotoxicity against B16 and secreted Th2 cytokines. Furthermore, the freshly isolated NK1.1+ TIL population exhibited a higher anti-B16 effect than did splenocytes. γδ T and αβ T cell populations dramatically inhibited the cytotoxicity of NK and NK T cells in an MHC Kb-dependent manner. Culture supernatant from γδ T and αβ T cell populations inhibited the proliferation of NK and NK T cell populations but did not affect their cytotoxicity, suggesting that the released Th2 cytokines are merely partly involved in the down-modulation of NK-lineage cells. NK1.1+ cells obtained from TIL of γδ T cell-depleted mice significantly lysed B16 cells compared with those from control mice. Finally, anti-Kb Fab mAb injected intralesionally at an early, but not at a late, stage of development of B16 melanoma inhibited tumor growth. These findings suggest that Th2-type γδ T and αβ T cells infiltrating in early B16 development inhibit the tumoricidal activity of NK-lineage cells using their class I molecules and partly their suppressive cytokines.