bluuebunny/crossref_metadata_2025 huggingface dataset:
Anchorage Dependence of Mitogen-Induced G1 to S Transition in Primary T Lymphocytes
Authors: Geginat J. , Bossi G. , Bender J. R. , Pardi R.
Published: December 2022

Anchorage dependence defines the cellular requirement for integrin-mediated adhesion to substrate to initiate DNA replication in response to growth factors. In this study we investigated whether normal T cells, which spend extended periods in a nonadherent state, show similar requirements for cell cycle progression in response to TCR stimulation. Resting primary T lymphocytes were induced to enter the cell cycle by TCR triggering, and leukocyte integrins were either engaged using purified ICAM-1 or inhibited with function-blocking mAbs. Our data indicate that leukocyte integrins complement TCR-driven mitogenic signals not as a result of their direct clustering but, rather, via integrin-dependent organization of the actin cytoskeleton. Leukocyte integrin-dependent reorganization of the actin cytoskeleton cooperates with the TCR to effect mitogen-activated protein kinase activation, but also represents a required late (4–8 h poststimulation) component in the mitogenic response of normal T cells. Prolonged leukocyte integrin-dependent spreading, in the context of intercellular contact, is a requisite for the production of the mitogenic cytokine IL-2, which, in turn, is involved in the induction of D3 cyclin and is primarily responsible for the decrease in the cyclin-dependent kinase inhibitor p27kip, resulting in retinoblastoma protein inactivation and S phase entry. Thus, T lymphocytes represent a peculiar case of anchorage dependence, in which signals conveyed by integrins act sequentially with the activating stimulus to effect a sustained production of the essential mitogenic cytokine.
Critical Role of Leukocyte Function-Associated Antigen-1 in Liver Accumulation of CD4+NKT Cells
Authors: Emoto Masashi , Mittrücker Hans-Willi , Schmits Rudolf , Mak Tak W. , Kaufmann Stefan H. E.
Published: December 2022

In contrast to peripheral lymphoid organs, a high percentage of T cells in the liver are CD4+NKT cells. We asked whether adhesion molecules play any role in the accumulation of CD4+NKT cells in the liver. Liver CD4+NKT cells expressed ICAM-1 and high levels of LFA-1. In the livers of LFA-1-deficient mice, the number of CD4+NKT cells was markedly decreased. This reduction was restricted to the liver, and no reduction was found in the other organs analyzed. In contrast, the number of liver CD4+NKT cells in ICAM-1-deficient mice was only marginally reduced. In a reciprocal radiation thymocyte reconstitution system with LFA-1-deficient and wild-type mice, LFA-1 expressed on liver cells other than CD4+NKT cells was required for an accumulation of CD4+NKT cells in the liver. These results demonstrate a crucial role for LFA-1 in the accumulation of CD4+NKT cells in the liver.
Dynamics and Requirements of T Cell Clonal Expansion In Vivo at the Single-Cell Level: Effector Function Is Linked to Proliferative Capacity
Authors: Gudmundsdottir Hrefna , Wells Andrew D. , Turka Laurence A.
Published: December 2022

The adoptive transfer of TCR-transgenic T cells into syngeneic recipients allows characterization of individual T cells during in vivo immune responses. However, the proliferative behavior of individual T cells and its relationship to effector and memory function has been difficult to define. Here, we used a fluorescent dye to dissect and quantify T cell proliferative dynamics in vivo. We find that the average Ag-specific CD4+ T cell that undergoes division in vivo generates >20 daughter cells. TCR and CD28 signals cooperatively determine the degree of primary clonal expansion by increasing both the proportion of Ag-specific T cells that divide and the number of rounds of division the responding T cells undergo. Nonetheless, despite optimal signaling, up to one-third of Ag-specific cells fail to divide even though they show phenotypic evidence of Ag encounter. Surprisingly, however, transgenic T cells maturing on a RAG-2−/− background exhibit a responder frequency of 95–98% in vivo, suggesting that maximal proliferative potential requires either a naive phenotype or allelic exclusion at the TCRα locus. Finally, studies reveal division cycle-dependent expression of markers of T cell differentiation, such as CD44, CD45RB, and CD62L, and show also that expression of the cytokines IFN-γ and IL-2 depends primarily on cell division rather than on receipt of costimulatory signals. These results provide a quantitative assessment of T cell proliferation in vivo and define the relationship between cell division and other parameters of the immune response including cytokine production, the availability of costimulation, and the capacity for memory.
CD45 Regulates Tyrosine Phosphorylation of CD22 and Its Association with the Protein Tyrosine Phosphatase SHP-1
Authors: Greer Susanna F. , Justement Louis B.
Published: December 2022

Cross-linking of CD45 induced capping and physical sequestration from CD22 leading to an increase in tyrosine phosphorylation of CD22 and SHP-1 recruitment. Additionally, CD22 isolated from a CD45-deficient B cell line exhibited increased basal/inducible tyrosine phosphorylation and enhanced recruitment of SHP-1 compared with CD22 isolated from CD45-positive parental cells. Subsequent experiments were performed to determine whether enhanced SHP-1 recruitment to CD22 is responsible for attenuation of receptor-mediated Ca2+ responses in CD45-deficient cells. Catalytically inactive SHP-1 expressed in CD45-deficient cells interacted with CD22 and decreased phosphatase activity in CD22 immunoprecipitates to levels that were comparable to those in CD45-positive cells. Expression of catalytically inactive SHP-1 restored intracellular mobilization of Ca2+ in response to MHC class II cross-linking, but did not affect B cell Ag receptor- or class II-mediated Ca2+ influx from the extracellular space. These results indicate that CD45 regulates tyrosine phosphorylation of CD22 and binding of SHP-1. The data further indicate that enhanced recruitment and activation of SHP-1 in CD45-deficient cells affect intracellular mobilization of Ca2+, but are not responsible for abrogation of receptor-mediated Ca2+ influx from the extracellular space.
Mice Deficient in CD4 T Cells Have Only Transiently Diminished Levels of IFN-γ, Yet Succumb to Tuberculosis
Authors: Caruso Amy Myers , Serbina Natalya , Klein Edwin , Triebold Karla , Bloom Barry R. , Flynn JoAnne L.
Published: December 2022

CD4 T cells are important in the protective immune response against tuberculosis. Two mouse models deficient in CD4 T cells were used to examine the mechanism by which these cells participate in protection against Mycobacterium tuberculosis challenge. Transgenic mice deficient in either MHC class II or CD4 molecules demonstrated increased susceptibility to M. tuberculosis, compared with wild-type mice. MHC class II−/− mice were more susceptible than CD4−/− mice, as measured by survival following M. tuberculosis challenge, but the relative resistance of CD4−/− mice did not appear to be due to increased numbers of CD4−8− (double-negative) T cells. Analysis of in vivo IFN-γ production in the lungs of infected mice revealed that both mutant mouse strains were only transiently impaired in their ability to produce IFN-γ following infection. At 2 wk postinfection, IFN-γ production, assessed by RT-PCR and intracellular cytokine staining, in the mutant mice was reduced by >50% compared with that in wild-type mice. However, by 4 wk postinfection, both mutant and wild-type mice had similar levels of IFN-γ mRNA and protein production. In CD4 T cell-deficient mice, IFN-γ production was due to CD8 T cells. Thus, the importance of IFN-γ production by CD4 T cells appears to be early in infection, lending support to the hypothesis that early events in M. tuberculosis infection are crucial determinants of the course of infection.
Immune-Stimulating Complexes Induce an IL-12-Dependent Cascade of Innate Immune Responses
Authors: Smith Rosemary E. , Donachie Anne M. , Grdic Dubravka , Lycke Nils , Mowat Allan McI.
Published: December 2022

The development of subunit vaccines requires the use of adjuvants that act by stimulating components of the innate immune response. Immune-stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A are potential vaccine vectors that induce a wide range of Ag-specific responses in vivo encompassing both humoral and CD4 and CD8 cell-mediated immune responses. ISCOMS are active by both parenteral and mucosal routes, but the basis for their adjuvant properties is unknown. Here we have investigated the ability of ISCOMS to recruit and activate innate immune responses as measured in peritoneal exudate cells. The i.p. injection of ISCOMS induced intense local inflammation, with early recruitment of neutrophils and mast cells followed by macrophages, dendritic cells, and lymphocytes. Many of the recruited cells had phenotypic evidence of activation and secreted a number of inflammatory mediators, including nitric oxide, reactive oxygen intermediates, IL-1, IL-6, IL-12, and IFN-γ. Of the factors that we investigated further only IL-12 appeared to be essential for the immunogenicity of ISCOMS, as IL-6- and inducible nitric oxide synthase knockout (KO) mice developed normal immune responses to OVA in ISCOMS, whereas these responses were markedly reduced in IL-12KO mice. The recruitment of peritoneal exudate cells following an injection of ISCOMS was impaired in IL-12KO mice, indicating a role for IL-12 in establishing the proinflammatory cascade. Thus, ISCOMS prime Ag-specific immune responses at least in part by activating IL-12-dependent aspects of the innate immune system.
Regulation of Inflammatory Responses by Oncostatin M
Authors: Wallace Philip M. , MacMaster John F. , Rouleau Katherine A. , Brown T. Joseph , Loy James K. , Donaldson Karen L. , Wahl Alan F.
Published: December 2022

Oncostatin M (OM) is a pleiotropic cytokine produced late in the activation cycle of T cells and macrophages. In vitro it shares properties with related proteins of the IL-6 family of cytokines; however, its in vivo properties and physiological function are as yet ill defined. We show that administration of OM inhibited bacterial LPS-induced production of TNF-α and lethality in a dose-dependent manner. Consistent with these findings, OM potently suppressed inflammation and tissue destruction in murine models of rheumatoid arthritis and multiple sclerosis. T cell function and Ab production were not impaired by OM treatment. Taken together these data indicate the activities of this cytokine in vivo are antiinflammatory without concordant immunosuppression.
IL-12, IFN-γ, and T Cell Proliferation to Measles in Immunized Infants
Authors: Gans Hayley A. , Maldonado Yvonne , Yasukawa Linda L. , Beeler Judy , Audet Susette , Rinki Mary M. , DeHovitz Ross , Arvin Ann M.
Published: December 2022

Measles infection in infants is associated with severe complications, and secondary infections are attributed to generalized immunosuppression. Measles binding to its monocyte receptor down-regulates IL-12 which is expected to diminish Th1-like cytokine responses, including IFN-γ. Whether young infants can be immunized effectively against measles is an important public health issue. We evaluated Ag-specific IL-12, IFN-γ, and T cell responses of infants at 6 (n = 60), 9 (n = 46), or 12 mo (n = 56) of age and 29 vaccinated adults. IL-12 and IFN-γ release by PBMC stimulated with measles Ag increased significantly after measles immunization in infants. IL-12 and IFN-γ concentrations were equivalent in younger and older infants, but IL-12 concentrations were significantly lower in infants than in adults (p = 0.04). IL-12 production by monocytes was down-regulated by measles; the addition of recombinant human IL-12 enhanced IFN-γ production by PBMC stimulated with measles Ag, but infant T cells released significantly less IFN-γ than adult T cells under this condition. Of particular interest, the presence of passive Abs to measles had no effect on the specific T cell proliferation or IFN-γ production after measles stimulation. Cellular immunity to measles infection and vaccination may be limited in infants compared with adults as a result of less effective IFN-γ and IL-12 production in response to measles Ags. These effects were not exaggerated in younger infants compared with effects in infants who were immunized at 12 mo. In summary, infant T cells were primed with measles Ag despite the presence of passive Abs, but their adaptive immune responses were limited compared with those of adults.
DNA Immunization with HIV-1tatMutated in thetransActivation Domain Induces Humoral and Cellular Immune Responses Against Wild-Type Tat
Authors: Caselli Elisabetta , Betti Monica , Grossi Maria Pia , Balboni Pier Giorgio , Rossi Cristina , Boarini Chiara , Cafaro Aurelio , Barbanti-Brodano Giuseppe , Ensoli Barbara , Caputo Antonella
Published: December 2022

Intramuscular immunization of mice with plasmids encoding two transdominant negative mutants of the HIV-1 Tat protein (Tat22 and Tat22/37) elicited a humoral response to wild-type Tat that is comparable to that induced by inoculation of wild-type tat DNA or Tat protein. The percentage of the responders and the Ab titers continued to increase after three additional DNA boosts and pretreatment with bupivacaine at the site of inoculation, without a significant difference (p > 0.05) among the three groups of mice immunized with mutant and wild-type tat genes. By utilizing synthetic peptides representing the amino acid sequence of Tat, one major B cell epitope was defined within the cysteine-rich domain of Tat. Anti-Tat IgG Abs directed against this epitope were found in mice immunized with all tat DNA constructs, whereas different Tat epitopes were detected in mice immunized with the Tat protein. Similarly, IgG2a was the predominant isotype in DNA-immunized mice, with both mutants and wild-type tat genes, as compared with protein immunization, which induced mostly IgG1 and IgG3. Sera from most immunized mice neutralized the effect of extracellular Tat in activating HIV-1 replication. A cellular response was also elicited as indicated by the proliferation of splenocytes when stimulated with wild-type Tat. These results indicate that the wild-type Tat Ag is recognized by Abs and T cells induced by DNA immunization with mutated tat genes, suggesting the possible use of these Tat transdominant mutants, lacking viral trans activation activity and capable of blocking wild-type Tat activity, in the development of an anti-HIV-1 vaccine.
Proteoglycan (Aggrecan)-Induced Arthritis in BALB/c Mice Is a Th1-Type Disease Regulated by Th2 Cytokines
Authors: Finnegan Alison , Mikecz Katalin , Tao Ping , Glant Tibor T.
Published: December 2022

In animal models of arthritis induced with Ags or infectious agents, disease severity correlates with a dominant Th1-type response characterized by a higher ratio of IFN-γ to IL-4. Analysis of BALB/c mice revealed a genetic predisposition toward developing CD4+ Th2-type responses. The bias toward an IL-4-dominant response in BALB/c mice protects mice from severe Lyme-induced arthritis and spontaneous autoimmune disease. Since BALB/c mice immunized with proteoglycan develop severe arthritis, we were interested in testing whether arthritis is associated with a Th2-type response and thus is different from other arthritic models. BALB/c mice immunized with proteoglycan generated a higher ratio of IFN-γ to IL-4 that peaks at the onset of arthritis. We investigated whether when Th1 cells were dominant, disease outcome could be modified with pharmacological amounts of Th2 cytokines. Treatment with IL-4 prevented disease and induced a switch from a Th1-type to a Th2-type response. Proinflammatory cytokine mRNA transcripts were reduced in joints of cytokine-treated mice. Th2 cytokine therapy at the time of maximum joint inflammation also suppressed symptoms of disease. Despite the predisposition of BALB/c mice to a Th2-type response, proteoglycan-induced arthritis is a Th1-type disease. The effectiveness of IL-4 treatment was particularly striking because in other models of arthritis, treatment in a similar manner with IL-4 was not sufficient to inhibit arthritis. The effective control of arthritis and the switch from a Th1 to Th2 response suggest that levels of endogenous IL-4 in BALB/c mice may increase their responsiveness to Th2 cytokine therapy.
Gamma-Irradiation Directly Affects the Formation of Coding Joints in SCID Cell Lines
Authors: Binnie Alexandra , Olson Stacy , Wu Gillian E. , Lewis Susanna M.
Published: December 2022

SCID mice have a defect in the catalytic subunit of the DNA-dependent protein kinase, causing increased sensitivity to ionizing radiation in all tissues and severely limiting the development of B and T cell lineages. SCID T and B cell precursors are unable to undergo normal V(D)J recombination: coding joint and signal joint products are less frequently formed and often will exhibit abnormal structural features. Paradoxically, irradiation of newborn SCID mice effects a limited rescue of T cell development. It is not known whether irradiation has a direct impact on the process of V(D)J joining, or whether irradiation of the thymus allows the outgrowth of rare recombinants. To investigate this issue, we sought to demonstrate an irradiation effect ex vivo. Here we have been able to reproducibly detect low-frequency coding joint products with V(D)J recombination reporter plasmids introduced into SCID cell lines. Exposure of B and T lineage cells to 100 cGy of gamma irradiation made no significant difference with respect to the number of coding joint and signal joint recombination products. However, in the absence of irradiation, the coding joints produced in SCID cells had high levels of P nucleotide insertion. With irradiation, markedly fewer P insertions were seen. The effect on coding joint structure is evident in a transient assay, in cultured cells, establishing that irradiation has an immediate impact on the process of V(D)J recombination. A specific proposal for how the DNA-dependent protein kinase catalytic subunit influences the opening of hairpin DNA intermediates during coding joint formation in V(D)J recombination is presented.
Early B Cell Factor Is an Activator of the B Lymphoid Kinase Promoter in Early B Cell Development
Authors: Åkerblad Peter , Sigvardsson Mikael
Published: December 2022

Early B cell factor (EBF) is a transcription factor suggested to be involved in the transcriptional control of several B cell restricted genes. EBF is also essential for B lymphocyte development because mice carrying a homologous disruption of the EBF gene lack mature B lymphocytes. This makes the identification of genetic targets for EBF important for the understanding of early B cell development. Examination of the nucleotide sequence of the B lymphoid kinase (Blk) promoter suggested the presence of an EBF binding site, and in vivo footprinting analysis showed that the site was protected from methylation in a pre-B cell line. EMSA indicated that recombinant and cellular EBF interact physically with this site; furthermore, transient transfections indicated that ectopic expression of EBF in nonlymphoid HeLa cells activate a Blk promoter-controlled reporter construct 9-fold. The defined EBF binding site was also important for the function of the Blk promoter in pre-B cells, because transient transfections of a reporter construct under the control of an EBF site-mutated Blk promoter displayed only 20–30% of the activity of the wild-type promoter. Furthermore, transient transfections in HeLa cells proposed that EBF and B cell-specific activator protein were able to cooperate in the activation of a Blk promoter-controlled reporter construct. These data indicate that EBF plays an important role in the regulation of the Blk promoter in early B cell development and that EBF and BSAP are capable to act in cooperation to induce a target gene.
Defects in the Generation of IFN-γ Are Overcome to Control Infection withLeishmania donovaniin CC Chemokine Receptor (CCR) 5-, Macrophage Inflammatory Protein-1α-, or CCR2-Deficient Mice
Authors: Sato Naoko , Kuziel William A. , Melby Peter C. , Reddick Robert Lee , Kostecki Vannessa , Zhao Weiguo , Maeda Nobuyo , Ahuja Sunil K. , Ahuja Seema S.
Published: December 2022

We investigated the immune responses in mice lacking CCR2, CCR5, or macrophage inflammatory protein-1α (MIP-1α), a ligand for CCR5, in two situations: following T cell stimulation or after challenge with Leishmania donovani, an intracellular microbe whose control is dependent on a Th1 immune response. Mice deficient in CCR5, MIP-1α, or CCR2 had reduced IFN-γ responses following ligation of the TCR. Reduced IFN-γ responses following PMA and ionomycin were also observed in CD8+ T cells of CCR5−/− and CCR2−/− mice. During the early phases of infection, all three knockout mice had low Ag-specific IFN-γ responses. However, this reduced IFN-γ response was overcome during a state of persistent Ag stimulation (chronic infection), and was not associated with an adverse parasitologic outcome in any of the gene-targeted mouse strains. To the contrary, during the late phase of infection, an exaggerated Ag-specific IFN-γ response was evident in CCR5−/− and MIP-1α−/− mice, and this correlated with an enhanced control of parasite replication. Although granuloma formation was abnormal in each of the knockout mice, there was no correlation between the number or architecture of the granulomas and parasite burden. Collectively, these findings indicate an important role for CCR5, MIP-1α, and CCR2 in granulomatous inflammation, and that CCR5 and MIP-1α, possibly acting through CCR5, might play a deleterious role in the outcome of chronic L. donovani infection. Our data also suggest that there might be cross-talk between TCR and chemokine receptor signaling pathways.
Development of Systemic Immunity to Glioblastoma Multiforme Using Tumor Cells Genetically Engineered to Express the Membrane-Associated Isoform of Macrophage Colony-Stimulating Factor
Authors: Graf Martin R. , Jadus Martin R. , Hiserodt John C. , Wepsic H. Terry , Granger Gale A.
Published: December 2022

We investigated the ability of Fischer rat T9 glioblastoma cells transduced with cDNA genes for the secreted (s) or membrane-associated (m) isoform of M-CSF to elicit an antitumor response when implanted into syngeneic animals. Intracranial (i.c.) implantation of 1 × 105 T9 cells expressing mM-CSF (T9/mM-CSF) resulted in 80% tumor rejection. Electron microscopy of the T9/mM-CSF tumor site, 2–4 days postimplantation, showed marked infiltration by macrophages, many of which were in physical contact with the T9/mM-CSF cells. Animals that rejected T9/mM-CSF cells were resistant to i.c. rechallenge with T9 cells, but not syngeneic MadB106 breast adenocarcinoma cells, suggesting that T9-specific immunity can be generated within the brain via the endogenous APCs. Intracranial injection of parental T9, vector control (T9/LXSN), or T9 cells secreting M-CSF (T9/sM-CSF) was 100% fatal. Subcutaneous injection of 1 × 107 T9/sM-CSF, T9/LXSN, or parental T9 cells resulted in progressive tumors. In contrast, T9/mM-CSF cells injected s.c. were destroyed in 7–10 days and animals developed systemic immunity to parental T9 cells. Passive transfer of CD3+ T cells from the spleens of immune rats into naive recipients transferred T9 glioma-specific immunity. In vitro, splenocytes from T9/mM-CSF-immunized rats specifically proliferated in response to various syngeneic glioma stimulator cells. However, only marginal T cell-mediated cytotoxicity was observed by these splenocytes in a CTL assay against T9 target cells, regardless of restimulation with T9 cells. Subcutaneous immunization with viable T9/mM-CSF cells was effective in eradicating i.c. T9 tumors.
Resistance of Rheumatoid Synovial Dendritic Cells to the Immunosuppressive Effects of IL-10
Authors: MacDonald Kelli P. A. , Pettit Allison R. , Quinn Christopher , Thomas Gregory J. , Thomas Ranjeny
Published: December 2022

IL-10 down-regulates the APC function of many dendritic cells (DC), including human peripheral blood (PB) DC. In rheumatoid arthritis (RA), synovial fluid (SF) DC express markers of differentiation and are effective APC despite abundant synovial IL-10. The regulation of DC responsiveness to IL-10 was therefore examined by comparing the effect of IL-10 on normal PB and RA SF DC. Whereas IL-10 down-modulated APC function and MHC class II and B7 expression of PB DC, IL-10 had no such effect on SF DC. Since SF DC have differentiated in vivo in the presence of proinflammatory cytokines, PB DC were cocultured in the presence of IL-10 and either GM-CSF, IL-1β, TNF-α, IL-6, or TGF-β. GM-CSF, IL-1β, and TNF-α were all able to restore APC function. Whereas the effects of IL-10 on PB DC were shown to be mediated by IL-10R1, neither PB nor RA SF DC constitutively expressed IL-10R1 mRNA or detectable surface protein. In contrast, IL-10R1 protein was demonstrated in PB and SF DC whole cell lysates, suggestive of predominant intracellular localization of the receptor. Thus, DC responsiveness to IL-10 may be regulated through modulation of cell surface IL-10R1 expression or signaling.
Differential Regulation of Epithelial-Derived C-C Chemokine Expression by IL-4 and the Glucocorticoid Budesonide
Authors: Stellato Cristiana , Matsukura Satoshi , Fal Andrzej , White John , Beck Lisa A. , Proud David , Schleimer Robert P.
Published: December 2022

Airway epithelial cells are a rich source of eosinophil-selective C-C chemokines. We investigated whether cytokines and the topical glucocorticoid budesonide differentially regulate RANTES, monocyte chemoattractant protein-4 (MCP-4), and eotaxin mRNA and protein expression in the human bronchial epithelial cell line BEAS-2B and in primary human bronchial epithelial cells by Northern blot analysis and ELISAs. Eotaxin and MCP-4 mRNA expression induced by TNF-α alone or in combination with IFN-γ was near-maximal after 1 h, peaked at 4 and 8 h, respectively, remained unchanged up to 24 h, and was protein synthesis independent. In contrast, RANTES mRNA was detectable only after 2 h and slowly increased to a peak at 24 h, and was protein synthesis dependent. Induction of eotaxin and MCP-4 mRNA showed a 10- to 100-fold greater sensitivity to TNF-α compared with RANTES mRNA. IL-4 and IFN-γ had selective effects on chemokine expression; IL-4 selectively up-regulated the expression of eotaxin and MCP-4 and potentiated TNF-α-induced eotaxin, while IFN-γ markedly potentiated only the TNF-α-induced expression of RANTES. Although budesonide inhibited the expression of chemokine mRNA to a variable extent, it effectively inhibited production of eotaxin and RANTES protein. Budesonide inhibited both RANTES- and eotaxin promoter-driven reporter gene activity. Budesonide also selectively accelerated the decay of eotaxin and MCP-4 mRNA. These results point to IL-4 as a possible mediator by which Th2 cells may induce selective production of C-C chemokines from epithelium and indicate that glucocorticoid inhibit chemokine expression through multiple mechanisms of action.
Transgenic Mice Expressing Different Levels of Soluble Platelet/Endothelial Cell Adhesion Molecule-IgG Display Distinct Inflammatory Phenotypes
Authors: Liao Fang , Schenkel Alan R. , Muller William A.
Published: December 2022

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), expressed on the surfaces of leukocytes and concentrated in the junctions between endothelial cells plays an important role in transendothelial migration of neutrophils and monocytes. Soluble recombinant PECAM-IgG injected i.v. into mice blocks acute leukocyte emigration by 80%. To study the role of PECAM in models of chronic inflammation, we generated transgenic mice constitutively expressing soluble full-length murine PECAM as an IgG chimera. Three founder lines expressed this transgene and constitutively secreted murine PECAM-IgG into the plasma where it was maintained at characteristic concentrations for each line. All mice had similar hematologic profiles to wild-type littermates and were healthy when maintained in the standard laboratory animal facility. Both the leukocytes and the endothelium of mice of all transgenic lines expressed the same levels of endogenous PECAM-1 as wild-type littermates. Similarly, there were no detectable differences in the expression of several other common leukocyte and endothelial cell adhesion molecules. Mice that produced moderate (10–20 μg/ml) concentrations of PECAM-IgG demonstrated a severely blunted acute inflammatory response, despite mobilizing appropriate numbers of circulating leukocytes. Surprisingly, mice that constitutively produced high (400–1000 μg/ml) concentrations of PECAM-IgG were unresponsive to its anti-inflammatory effects. This is the first demonstration that a soluble form of a cell adhesion molecule can be stably expressed and retain efficacy in vivo over prolonged periods. This approach is applicable to many other extracellular molecules. However, the plasma concentrations of such constitutively produced inhibitors may greatly influence the resulting phenotype.
Substance P Activates Coincident NF-AT- and NF-κB-Dependent Adhesion Molecule Gene Expression in Microvascular Endothelial Cells Through Intracellular Calcium Mobilization
Authors: Quinlan Kimberly L. , Naik Shubhada M. , Cannon Georgetta , Armstrong Cheryl A. , Bunnett Nigel W. , Ansel John C. , Caughman S. Wright
Published: December 2022

Upon stimulation, cutaneous sensory nerves release neuropeptides such as substance P (SP), which modulate responses in the skin by activating a number of target cells via neurokinin receptors. We have demonstrated that SP preferentially binds to the NK-1R on human dermal microvascular cells, resulting in increased intracellular Ca2+ and induction of ICAM-1 and VCAM-1 expression. In the current studies, we identify specific elements in the regulatory regions of ICAM-1 and VCAM-1 genes as necessary and sufficient for SP-dependent transcriptional activation. SP treatment of human dermal microvascular endothelial cells leads to coincident activation and binding of the transcription factor NF-AT to the −191/−170 region of the ICAM-1 gene (a region bound by activated p65/p65 homodimers in response to TNF-α), and NF-κB (p65/p50) to tandem NF-κB binding sites at −76/−52 of the VCAM-1 gene. The SP-elicited intracellular Ca2+ signal was required for activation and subsequent binding of both NF-AT and NF-κB. The transacting factor induction by SP was specific, since a selective NK-1R antagonist blocked SP activation and subsequent NF-AT and NF-κB activation and binding. These data demonstrate coincident activation of NF-AT and NF-κB via SP-induced intracellular Ca2+ mobilization and indicate a crucial role for neuropeptides in modulating localized cutaneous inflammatory responses.
CD4+ Tumor-Infiltrating Lymphocytes in Cervical Cancer Recognize HLA-DR-Restricted Peptides Provided by Human Papillomavirus-E7
Authors: Höhn Hanni , Pilch Henryk , Günzel Susanne , Neukirch Claudia , Hilmes Christine , Kaufmann Andreas , Seliger Barbara , Maeurer Markus J.
Published: December 2022

Human papillomavirus (HPV)-encoded proteins may provide targets for CD8+ or CD4+ T lymphocytes infiltrating into cervical cancer. We established an MHC class II-restricted CD4+ T cell line from a patient with cervical cancer that recognizes autologous (HPV35+, HPV59+) cervical cancer cells and the HLA-DR4-matched cervical cancer cell line Me180 (HPV68+) as determined by TNF-α secretion. Expression of different HPV-E7 genes in autologous B cells revealed that this T cell line defines a DR4-presented T cell epitope that is shared among the E7 genes of HPV59 and HPV68. MHC class II-presented peptides may be implemented to augment T cell responses directed against autologous tumor cells, particularly if cancer cells lack MHC class I expression, which is a frequent event in the evolution of cervical cancer.
Identification of Pathogenic T Cells in Patients with Beryllium-Induced Lung Disease
Authors: Fontenot Andrew P. , Falta Michael T. , Freed Brian M. , Newman Lee S. , Kotzin Brian L.
Published: December 2022

Chronic beryllium disease (CBD) is caused by beryllium exposure and is characterized by granulomatous inflammation with accumulation of CD4+ T cells in the lung. We analyzed TCR β-chain and α-chain genes expressed by these CD4+ T cells. In the lungs of individual patients, as well as among four of five CBD patients studied, different oligoclonal expansions within the Vβ3 subset were found to express homologous or even identical CDR3 amino acid sequences. These related expansions were specific for CBD patients, were compartmentalized to lung, and persisted at high frequency in patients with active disease. Limiting dilution cloning and analysis of coexpressed TCR α-chain genes confirmed that these TCRs were selectively expanded by a common Ag involving beryllium. Overall, homologous TCR β- and α-chains showed identical V regions and invariant charged residues within the CDR3 but considerable variability in TCRJ usage. Remarkably, CBD patients expressing nearly identical TCRs did not share common HLA-DRB1 or DQ alleles. These results implicate particular CD4+ cells in the pathogenesis of CBD and provide insight into how beryllium is recognized in human disease.
Ig λ and Heavy Chain Gene Usage in Early Untreated Systemic Lupus Erythematosus Suggests Intensive B Cell Stimulation
Authors: Dörner Thomas , Farner Nancy L. , Lipsky Peter E.
Published: December 2022

To determine the distribution of Vλ and Jλ as well as VH and JH gene usage in a patient with systemic lupus erythematosus (SLE), productive and nonproductive VJ and V(D)J rearrangements were amplified from individual peripheral CD19+ B cells and were analyzed. No differences in the Vλ and Jλ or the VH and JH gene usage in the nonproductive gene repertoire of this SLE patient were found compared with the distribution of genes found in normal adults, whereas marked skewing of both Vλ and VH was noted among the productive rearrangements. The distribution of productive Vλ rearrangements was skewed, with significantly greater representation of the Jλ distal cluster C Vλ genes and the Vλ distal Jλ7 element, consistent with the possibility that there was receptor editing of the Vλ locus in this patient. Significant bias in VH gene usage was also noted with VH3 family members dominating the peripheral B cell repertoire of the SLE patient (83%) compared with that found in normal subjects (55%; p < 0.001). Notably, a clone of B cells employing the VH3-11 gene for the heavy chain and the Vλ1G segment for the light chain was detected. These data are most consistent with the conclusion that extreme B cell overactivity drives the initial stages of SLE leading to remarkable changes in the peripheral V gene usage that may underlie on fail to prevent the emergence of autoimmunity.
Anti-Human Cardiac Myosin Autoantibodies in Kawasaki Syndrome
Authors: Cunningham Madeleine W. , Meissner H. Cody , Heuser Janet S. , Pietra Biagio A. , Kurahara David K. , Leung2 Donald Y. M.
Published: December 2022

Kawasaki syndrome (KS) is the major cause of acquired heart disease in children. Although acute myocarditis is observed in most patients with KS, its pathogenesis is unknown. Because antimyosin autoantibodies are present in autoimmune myocarditis and rheumatic carditis, the purpose of the current study was to determine whether anticardiac myosin Abs might be present during the acute stage of KS. Sera from KS patients as well as age-matched febrile controls and normal adults were compared for reactivity with human cardiac myosin in ELISAs and Western blot assays. A total of 5 of 13 KS sera, as compared with 5 of 8 acute rheumatic fever sera, contained Ab titers to human cardiac myosin that were significantly higher than those found in control sera. Both cardiac and skeletal myosins were recognized in the ELISA by KS sera, although stronger reactivity was observed to human cardiac myosin. Only IgM antimyosin Abs from KS sera were significantly elevated relative to control sera. KS sera containing antimyosin Abs recognized synthetic peptides from the light meromyosin region of the human cardiac myosin molecule and had a different pattern of reactivity than acute rheumatic fever sera, further supporting the association of antimyosin Ab with KS. These Abs may contribute to the pathogenesis of acute myocarditis found in patients with KS.
Autoantibodies to the Extracellular Matrix Microfibrillar Protein, Fibrillin-1, in Patients with Scleroderma and Other Connective Tissue Diseases
Authors: Tan Filemon K. , Arnett Frank C. , Antohi Stephan , Saito Shinichiro , Mirarchi Adriana , Spiera Harry , Sasaki Takeshi , Shoichi Ozaki , Takeuchi Ken , Pandey Janardan P. , Silver Richard M. , LeRoy Carwile , Postlethwaite Arnold E. , Bona Constantin A.
Published: December 2022

A duplication in the fibrillin-1 gene has been implicated as the cause of the tight skin 1 (tsk1) phenotype, an animal model of scleroderma or systemic sclerosis (SSc). In addition to the production of abnormal fibrillin-1 protein, the tsk1 mouse also produces autoantibodies to fibrillin-1. Among a population of Choctaw Native Americans with the highest prevalence of SSc yet described, a chromosome 15q haplotype containing the fibrillin-1 gene has been strongly associated with SSc. With a recombinant human fibrillin-1 protein, autoantibodies to fibrillin-1 were detected in the sera of Native American SSc patients that correlated significantly with disease. Abs to fibrillin-1 also were detected in sera from Japanese, Caucasian, and African-American SSc patients. Compared with other ethnic groups, Japanese and Native American SSc patients had significantly higher frequencies of anti-fibrillin-1 Abs. Sera from patients with diffuse SSc, calcinosis, Raynaud’s, esophageal dysmotility, sclerodactyly, and telangiectasias syndrome and mixed connective tissue disease also had significantly higher frequencies of anti-fibrillin-1 Abs than sera from controls or patients with other non-SSc connective tissue diseases (lupus, rheumatoid arthritis, and Sjögren’s syndrome). Ab specificity for fibrillin-1 was demonstrated by the lack of binding to a panel of other purified autoantigens. The results presented demonstrate for the first time the presence of high levels of anti-fibrillin-1 Abs in a significant portion of patients with SSc.
Retinal Expression of a Neo-Self Antigen, β-Galactosidase, Is Not Tolerogenic and Creates a Target for Autoimmune Uveoretinitis
Authors: Gregerson Dale S. , Torseth John W. , McPherson Scott W. , Roberts Josh P. , Shinohara Toshimichi , Zack Donald J.
Published: December 2022

Recent studies revealing active mechanisms of immune privilege in neural tissues have diminished the putative role of passive tolerance. To examine the significance of Ag localization in the retina on immune privilege, the immune responses of transgenic mice expressing high and low levels of β-galactosidase (β-gal) in the photoreceptor cells of the retina were compared with those of normal mice and those of mice expressing moderate levels of β-gal systemically. Immunization with β-gal induced experimental autoimmune uveoretinitis indistinguishable from that induced by known photoreceptor cell autoantigens, including destruction of photoreceptor cells, in transgenic mice with high level retinal expression. Retinal expression had no apparent effect on the immune responses to β-gal, showing that tolerance was not elicited by levels of retinal β-gal sufficient to serve as a target for autoimmune disease. Mice with systemic expression exhibited reduced lymphoproliferative responses following immunization with β-gal and did not develop autoimmune disease. T cells prepared from normal mice immunized with β-gal transferred experimental autoimmune uveoretinitis to the transgenic mice with high level retinal β-gal expression, but no disease was found in mice with systemic transgene expression under these conditions. The results of our experiments are most consistent with sequestration being the primary mechanism of retinal immune privilege. The results also show that β-gal can serve as an immunopathogenic neural autoantigen, and that T cells raised by immunization of normal mice with a foreign Ag can be immunopathogenic in certain transgenic recipients.
Resistance of Crohn’s Disease T Cells to Multiple Apoptotic Signals Is Associated with a Bcl-2/Bax Mucosal Imbalance
Authors: Ina Kenji , Itoh Jugoh , Fukushima Kouhei , Kusugami Kazuo , Yamaguchi Takeo , Kyokane Kazuhiro , Imada Akira , Binion David G. , Musso Alessandro , West Gail A. , Dobrea George M. , McCormick Thomas S. , Lapetina Eduardo G. , Levine Alan D. , Ottaway Clifford A. , Fiocchi Claudio
Published: December 2022

Crohn’s disease (CD) is a condition characterized by excessive numbers of activated T cells in the mucosa. We investigated whether a defect in apoptosis could prolong T cell survival and contribute to their accumulation in the mucosa. Apoptotic, Bcl-2+, and Bax+ cells in tissue sections were detected by the TUNEL method and immunohistochemistry. T cell apoptosis was induced by IL-2 deprivation, Fas Ag ligation, and exposure to TNF-α and nitric oxide. TUNEL+ leukocytes were few in control, CD, and ulcerative colitis (UC) mucosa, with occasional CD68+ and myeloperoxidase+, but no CD45RO+, apoptotic cells. Compared with control and UC, CD T cells grew remarkably more in response to IL-2 and were significantly more resistant to IL-2 deprivation-induced apoptosis. CD T cells were also more resistant to Fas- and nitric oxide-mediated apoptosis, whereas TNF-α failed to induce cell death in all groups. Compared with control, CD mucosa contained similar numbers of Bcl-2+, but fewer Bax+, cells, while UC mucosa contained fewer Bcl-2+, but more Bax+, cells. Hence, the Bcl-2/Bax ratio was significantly higher in CD and lower in UC. These results indicate that CD may represent a disorder where the rate of T cell proliferation exceeds that of cell death. Insufficient T cell apoptosis may interfere with clonal deletion and maintenance of tolerance, and result in inappropriate T cell accumulation contributing to chronic inflammation.
Differential CD3ζ Phosphorylation Is Not Required for the Induction of T Cell Antagonism by Altered Peptide Ligands
Authors: Liu Haiyan , Vignali Dario A. A.
Published: December 2022

T cells recognize foreign Ags in the form of short peptides bound to MHC molecules. Ligation of the TCR:CD3 complex gives rise to the generation of two tyrosine-phosphorylated forms of the CD3 ζ-chain, pp21 and pp23. Replacement of residues in MHC-bound peptides that alter its recognition by the TCR can generate altered peptide ligands (APL) that antagonize T cell responses to the original agonist peptide, leading to altered T cell function and anergy. This biological process has been linked to differential CD3ζ phosphorylation and generation of only the pp21 phospho-species. Here, we show that T cells expressing CD3ζ mutants, which cannot be phosphorylated, exhibit a 5-fold reduction in IL-2 production and a 30-fold reduction in sensitivity following stimulation with an agonist peptide. However, these T cells are still strongly antagonized by APL. These data demonstrate that: 1) the threshold required for an APL to block a response is much lower than for an agonist peptide to induce a response, 2) CD3ζ is required for full agonist but not antagonist responses, and 3) differential CD3ζ phosphorylation is not a prerequisite for T cell antagonism.
Differential Induction of Cytokine Genes and Activation of Mitogen-Activated Protein Kinase Family by Soluble CD40 Ligand and TNF in a Human Follicular Dendritic Cell Line
Authors: Park Sun-Mi , Kim Han-Soo , Choe Jongseon , Lee Tae H.
Published: December 2022

Follicular dendritic cells (FDC)3 play crucial roles in germinal center (GC) formation and differentiation of GC B cells. Many aspects of FDC function are influenced by contact with B or T cells, and by cytokines produced in the GC, which involve stimulation of CD40 and TNF-α receptors on FDC. In this study, using an established FDC line, HK cells, we compared the effects of CD40 and TNF receptor triggering on cytokine induction and activation of mitogen-activated protein kinase family. We show that HK cells spontaneously produced IL-6, M-CSF, and G-CSF mRNA. Both the soluble form of CD40 ligand (sCD40L) and TNF increased the level of M-CSF and G-CSF mRNA. While TNF strongly induced IL-6 mRNA, its expression was not affected by sCD40L treatment, differing from the strong IL-6 induction in other cell types upon CD40 stimulation. In addition, sCD40L treatment resulted in activation of extracellular signal-related kinase 1 and 2 (ERK1/2) and p38 without significant increase in c-Jun N-terminal kinase (JNK) activity. Lack of JNK activation differs in that most B cells respond to CD40 stimulation by inducing JNK activity strongly, suggesting distinct characteristics of CD40 signaling in FDC. Compared with the effects of sCD40L, TNF was capable of inducing JNK activity in addition to the activation of ERK1/2 and p38. Furthermore, the proximal signaling elements activated by TNF differed from those activated by sCD40L, in that TNF did not require PMA-sensitive protein kinase C isoforms in the activation of ERK and p38, whereas sCD40L did. However, signals activated by these stimuli converged on cytokine gene expression in a synergistic manner, which may have implication in augmenting FDC function during GC reaction.
Signaling Events Induced by Lipopolysaccharide-Activated Toll-Like Receptor 2
Authors: Yang Ruey-Bing , Mark Melanie R. , Gurney Austin L. , Godowski Paul J.
Published: December 2022

Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-κB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-κB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-κB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants.