bluuebunny/crossref_metadata_2025 huggingface dataset:
The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor.
Authors: Gumperz J E , Litwin V , Phillips J H , Lanier L L , Parham P
Published: June 2004

Although inhibition of natural killer (NK) cell-mediated lysis by the class I HLA molecules of target cells is an established phenomenon, knowledge of the features of class I molecules which induce this effect remains rudimentary. Using class I alleles HLA-B*1502 and B*1513 which differ only at residues 77-83 which define the Bw4 and Bw6 serological epitopes, we tested the hypothesis that the presence of the Bw4 epitope on class I molecules determines recognition by NKB1+ NK cells. HLA-B*1513 possesses the Bw4 epitope, whereas B*1502 has the Bw6 epitope. Lysis by NKB1+ NK cell clones of transfected target cells expressing B*1513 as the only HLA-A, -B, or -C molecule was inhibited, whereas killing of transfectants expressing B*1502 was not. Addition of an an anti-NKB1 monoclonal antibody reconstituted lysis of the targets expressing B*1513, but did not affect killing of targets bearing B*1502. The inhibitory effect of B*1513 could be similarly prevented by the addition of an anti-class I monoclonal antibody. These results show that the presence of the Bw4 epitope influences recognition of HLA-B molecules by NK cells that express NKB1, and suggest that the NKB1 molecule may act as a receptor for Bw4+ HLA-B alleles. Sequences outside of the Bw4 region must also affect recognition by NKB1+ NK cells, because lysis of transfectants expressing HLA-A*2403 or A*2501, which possess the Bw4 epitope but are in other ways substantially different from HLA-B molecules, was not increased by addition of the anti-NKB1 antibody. Asparagine 86, the single site of N-linked glycosylation on class I molecules, is in close proximity to the Bw4/Bw6 region. The glycosylation site of the Bw4-positive molecule B*5801 was mutated, and the mutant molecules tested for inhibition of NKB1+ NK cells. Inhibition that could be reversed by addition of the anti-NKB1 monoclonal antibody was observed, showing the presence of the carbohydrate moiety is not essential for class I recognition by NKB1+ NK cell clones.
Role of Rel-related factors in control of c-myc gene transcription in receptor-mediated apoptosis of the murine B cell WEHI 231 line.
Authors: Lee H , Arsura M , Wu M , Duyao M , Buckler A J , Sonenshein G E
Published: June 2004

Treatment of immature murine B lymphocytes with an antiserum against their surface immunoglobulin (sIg)M results in cell death via apoptosis. The WEHI 231 B cell line (IgM, kappa) has been used extensively as a model for this anti-Ig receptor-mediated apoptosis. Anti-sIg treatment of WEHI 231 cells causes an early, transient increase in the levels of c-myc messenger RNA and gene transcription, followed by a rapid decline below control values. Given the evidence for a role of the c-myc gene in promoting apoptosis, we have characterized the nature and kinetics of changes in the binding of Rel-related factors, which modulate c-myc promoter activity. In exponentially growing WEHI 231 cells, multiple Rel-related binding activities were detectable. The major binding species was identified as p50/c-Rel heterodimers; only minor amounts of nuclear factor kappa B (NF-kappa B) (p50/p65) were detectable. Cotransfection of an inhibitor of NF-kappa B (I kappa B)-alpha expression vector reduced c-myc-promoter/upstream/exon1-CAT reporter construct activity, indicating the role of Rel factor binding in c-myc basal expression in these cells. Treatment with anti-sIg resulted in a rapid transient increase in the rate of c-myc gene transcription and in the binding of Rel factors. At later times, formation of p50 homodimer complexes occurred. In cotransfection analysis, p65 and c-Rel expression potently and modestly transactivated the c-myc promoter, respectively, whereas, overexpression of the p50 subunit caused a significant drop in its activity. The role of activation of Rel-family binding was demonstrated directly upon addition of the antioxidant pyrrolidinedithiocarbamate, which inhibited the anti-sIg-mediated activation of the endogenous c-myc gene. Similarly, induction after anti-sIg treatment of a transfected c-myc promoter was abrogated upon cotransfection of an I kappa B-alpha expression vector. These results implicate the Rel-family in Ig receptor-mediated signals controlling the activation of c-myc gene transcription in WEHI 231 cells, and suggest a role for this family in apoptosis of this line, which is mediated through a c-myc signaling pathway.
P-selectin and vascular cell adhesion molecule 1 mediate rolling and arrest, respectively, of CD4+ T lymphocytes on tumor necrosis factor α-activated vascular endothelium under flow.
Authors: Luscinskas F W , Ding H , Lichtman A H
Published: June 2004

This report examines the adhesive interactions of human CD4+ T lymphocytes with tumor necrosis factor alpha-activated human endothelial cell monolayers in an in vitro model that mimics microcirculatory flow conditions. Resting CD4+ T cell interactions with activated endothelium consisted of initial attachment followed by rolling, stable arrest, and then spreading and transendothelial migration. P-selectin, but not E-, or L-selectin, mediated most of this initial contact and rolling, whereas beta 1-integrins (alpha 4 beta 1), interacting with endothelial-expressed vascular cell adhesion molecule 1, participated in rolling and mediated stable arrest. In contrast, beta 2-integrins were primarily involved in spreading and transmigration. These findings highlight an important role for P-selectin and suggest discrete functions for beta 1- and beta 2-integrins during lymphocyte recruitment to sites of immune-mediated inflammation.
Monocyte migration to arthritis in the rat utilizes both CD11/CD18 and very late activation antigen 4 integrin mechanisms.
Authors: Issekutz A C , Issekutz T B
Published: June 2004

In human and experimental models of arthritis, blood monocytes migrate into the inflamed synovium and joint space. The mechanisms required for monocyte migration across the vascular endothelium in joints is poorly defined. Radiolabeled rat blood monocytes were used to measure monocyte migration to the inflamed joints of rats with adjuvant arthritis, and the role of monocyte adhesion molecules was analyzed. Monocyte accumulation in the inflamed joints was maximal 14-21 d after immunization with adjuvant, when arthritis had fully developed. Blocking mAbs to lymphocyte function-associated antigen 1 (LFA-1), Mac-1, and very late activation antigen 4 (VLA-4) were used to evaluate the role of these integrins in the migration. Migration to the joints was not inhibited by treatment of the animals with mAb to LFA-1, Mac-1, or VLA-4 alone, and was partially (50%) inhibited in only the most arthritic joint, the talar joint, by the combination of mAb to LFA-1 plus Mac-1. In contrast, this combination inhibited migration to dermal inflammation induced by C5ades Arg, endotoxin, tumor necrosis factor alpha, and polyinosine-cytosine by 60-70%. When mAbs to LFA-1 and VLA-4 were combined, migration to all the inflamed joints was strongly inhibited (80-98%, depending on the joint). Treatment with the combination of the three mAbs to LFA-1, Mac-1, and VLA-4 completely eliminated monocyte migration to all joints and dermal inflammation. The results show that 51Cr blood monocytes can be used to quantify monocyte migration to arthritic joints in the rat. LFA-1 alone or VLA-4 alone is sufficient to mediate most of this migration, and either LFA-1 or VLA-4 is required for monocyte migration to joint inflammation. These results indicate that both the VLA-4 and LFA-1 integrins should be therapeutic targets for suppression of monocyte infiltration of joints in arthritis.
A metalloprotease inhibitor blocks shedding of the 80-kD TNF receptor and TNF processing in T lymphocytes.
Authors: Crowe P D , Walter B N , Mohler K M , Otten-Evans C , Black R A , Ware C F
Published: June 2004

TNF is synthesized as a 26-kD membrane-anchored precursor and is proteolytically processed at the cell surface to yield the mature secreted 17-kD polypeptide. The 80-kD tumor necrosis factor (TNF) receptor (TNFR80) is also proteolytically cleaved at the cell surface (shed), releasing a soluble ligand-binding receptor fragment. Since processing of TNF and TNFR80 occurs concurrently in activated T cells, we asked whether a common protease may be involved. Here, we present evidence that a recently described inhibitor of TNF processing N-(D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl)L- 3-(2'naphthyl)- alanyl-L-alanine, 2-aminoethyl amide (TAPI) also blocks shedding of TNFR80, suggesting that these processes may be coordinately regulated during T cell activation. In addition, studies of murine fibroblasts transfected with human TNFR80, or a cytoplasmic deletion form of TNFR80, reveal that inhibition of TNFR80 shedding by TAPI is independent of receptor phosphorylation and does not require the receptor cytoplasmic domain.
Calcium/calmodulin-dependent protein kinase II downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells.
Authors: Hama N , Paliogianni F , Fessler B J , Boumpas D T
Published: June 2004

Engagement of the T cell receptor for antigen activates phospholipase C resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of protein kinase C (PKC). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM-K II), as well as calcineurin, a type 2B protein phosphatase. Recent studies have identified calcineurin as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of CaM-K II remains unknown. We have used mutants of these kinases and phosphatases (gamma B*CaM-K and delta CaM-AI, respectively) to explore their relative role in cytokine gene transcription and their interactions with PKC-dependent signaling systems. gamma B*CaM-K and delta CaM-AI, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of gamma B*CaM-K with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of CaM-K II on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, delta CaM-AI superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, gamma B*CaM-K inhibited the induction of the IL-2 promoter by delta CaM-AI. Similar results were obtained when a construct containing the IL-4 promoter also was used. gamma B*CaM-K also downregulated the activation of AP-1 in response to transfection with a constitutively active mutant of PKC or stimulation with PMA. These results suggest that CaM-K II may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC-dependent signaling systems.
Transgenic expression of interleukin 7 restores T cell populations in nude mice.
Authors: Rich B E , Leder P
Published: June 2004

The thymic lesion of the nude mouse causes a profound block in T cell development. The failure of most T cells to mature in nude mice is likely to reflect a requirement for signals elaborated in the normal thymus. Interleukin 7 (IL-7), a lymphokine that is normally expressed in the thymus and has been implicated in T cell maturation, might be central to this process. To test this possibility, we introduced a transgene directing lymphoid expression of IL-7 into nude mice and found that it substantially alleviates the block in T cell maturation caused by the thymic defect. IL-7 transgenic nude mice have increased numbers of peripheral cells expressing the T cell marker Thy-1, the T cell antigen receptor complex, and the co-receptors CD4 and CD8. The IL-7 transgene also restores T cell-specific proliferation and activation responses to the peripheral cells of transgene-rescued nude mice. Such findings point toward a fundamental role for IL-7 in the thymic maturation of T cells.
Virus-specific CD8+ cells can switch to interleukin 5 production and induce airway eosinophilia.
Authors: Coyle A J , Erard F , Bertrand C , Walti S , Pircher H , Le Gros G
Published: June 2004

Virus infections of the lung are thought to predispose individuals to asthma, a disease characterized by eosinophil infiltration of the airways. CD8+ T cells are an important part of the host response to virus infection, however, they have no reported role in eosinophil recruitment. We developed a mouse model of virus peptide-stimulated CD8+ T cell immune responses in the lung. We found that bystander CD4+ T helper cell type 2 immune responses to ovalbumin switched the virus peptide-specific CD8+ T cells in the lung to interleukin (IL) 5 production. Furthermore, when such IL-5-producing CD8 T cells were challenged via the airways with virus peptide, a significant eosinophil infiltration was induced. In vitro studies indicated that IL-4 could switch the virus-specific CD8+ T cells to IL-5 production. These results could explain the link between virus infection and acute exacerbation of asthma and, perhaps more importantly, they indicate an IL-4-dependent mechanism that would impair CD8+ T cell responses and delay viral clearance from the host.
Fas-mediated cytotoxicity by freshly isolated natural killer cells.
Authors: Arase H , Arase N , Saito T
Published: June 2004

The expression of Fas ligand on natural killer (NK) cells and Fas-mediated cytotoxicity by NK cells was investigated. Fas ligand mRNA was expressed in freshly isolated NK cells but not in T cells. Furthermore, the Fas ligand was detected on the cell surface of NK cells by staining with soluble Fas molecule. We analyzed the cytolytic activity of NK cells against thymocyte targets from normal and lpr mice, and found that the NK cells killed thymocytes from normal mice but not from lpr mice. On the other hand, splenic T cells did not show any cytotoxicity against either of the thymocyte targets. Similarly, NK cells exhibited cytotoxicity against transfectants expressing Fas antigen but not against parental cells or transfectants expressing a mutant Fas antigen with deleted cytoplasmic region. These results demonstrated that NK cells express Fas ligand and possess the capability of killing target cells expressing Fas antigen on their surface. This finding suggests that NK cells play an important role by eliminating Fas-expressing cells either constitutively or inducibly in peripheral lymphoid organs.
γ/δ T lymphocytes express CD40 ligand and induce isotype switching in B lymphocytes.
Authors: Horner A A , Jabara H , Ramesh N , Geha R S
Published: June 2004

T cells expressing gamma/delta T cell receptors home to epithelial tissue and may play a role in immunity to infectious agents and foreign antigens. In an effort to understand the role of gamma/delta T cells in directing B cell responses, we investigated the capacity of human gamma/delta T cells to express CD40 ligand (CD40L) and to drive immunoglobulin (Ig) isotype switching in B cells. A multiple step purification procedure resulted in the recovery of highly pure populations of peripheral blood CD4-CD8- gamma/delta T cells. Neither CD40L surface expression nor CD40L mRNA were detected in unstimulated gamma/delta T cells. Stimulation with phorbol ester and ionomycin induced CD40L mRNA and surface CD40L expression by gamma/delta T cells. Both the percentage of CD40L+ cells and the cell surface density of CD40L were significantly lower in gamma/delta T cells compared to unselected T cells. We further demonstrated that in the presence of neutralizing monoclonal antibody to interferon gamma (IFN-gamma), gamma/delta T cells could induce IgE synthesis in B cells, albeit to a lesser extent than unselected T cells. Furthermore, IgE synthesis driven by gamma/delta T cells was inhibited by monoclonal antibody to CD40L. These observations demonstrate that activated gamma/delta T cells express CD40L and can induce isotype switching in B cells.
Chemoattraction of human blood T lymphocytes by interleukin-15.
Authors: Wilkinson P C , Liew F Y
Published: June 2004

Recombinant interleukin (IL)-15, derived from a simian kidney epithelial cell line, is a chemoattractant for human blood T lymphocytes judged by its ability to increase the proportion of cells in polarized morphology, to stimulate invasion of collagen gels containing IL-15, and to increase the proportion of locomotor cells observed by time-lapse videorecording. The ability of lymphocytes to respond was partly, but not completely, inhibited by pretreatment with anti-IL-2 receptor beta-chain. The activity of IL-15 was completely abolished by preincubation with aIL-15 but unaffected by preincubation with aIL-2. No response of monocytes, neutrophils, or B lymphocytes to IL-15 was observed.
Role of interleukin 10 in the B lymphocyte hyperactivity and autoantibody production of human systemic lupus erythematosus.
Authors: Llorente L , Zou W , Levy Y , Richaud-Patin Y , Wijdenes J , Alcocer-Varela J , Morel-Fourrier B , Brouet J C , Alarcon-Segovia D , Galanaud P , Emilie D
Published: June 2004

Interleukin-10 (IL-10) is produced at a high level by B lymphocytes and monocytes of patients with systemic lupus erythematosus (SLE). In the present work, we analyzed whether this increased production of IL-10 contributed to the abnormal production of immunoglobulins (Ig) and of autoantibodies in SLE. The role of IL-10 was compared with that of IL-6, another cytokine suspected to play a role in these abnormalities. The spontaneous in vitro production of IgM, IgG, and IgA by peripheral blood mononuclear cells from SLE patients was weakly increased by recombinant IL (rIL)-6, but strongly by rIL-10. This production was not significantly affected by an anti-IL-6 mAb but was decreased by an anti-IL-10 mAb. We then tested the in vivo effect of these antibodies in severe combined immunodeficiency mice injected with PBMC from SLE patients. The anti-IL-6 mAb did not significantly affect the serum concentration of total human IgG and of anti-double-stranded DNA IgG in the mice. In contrast, the anti-IL-10 mAb strongly inhibited the production of autoantibodies, and, to a lesser extent, that of total human IgG. These results indicate that the Ig production by SLE B lymphocytes is largely IL-10 dependent, and that the increased production of IL-10 by SLE B lymphocytes and monocytes may represent a critical mechanism in the emergence of the autoimmune manifestations of the disease.
T cell and non-T cell compartments can independently determine resistance to Leishmania major.
Authors: Shankar A H , Titus R G
Published: June 2004

In experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm), the cellular determinants governing development of protective or exacerbative T cells are not well understood. We, therefore, attempted to determine the influence of T cell and non-T cell compartments on disease outcome. To this end, T cell chimeric mice were constructed using adult thymectomized lethally irradiated, bone marrow-reconstituted (ATXBM) animals of genetically resistant, C57BL/6, or susceptible, BALB/c, backgrounds. These hosts were engrafted with naive T cell populations from H-2-congenic susceptible, BALB.B6-H-2b, or resistant, C57BL/6.C-H-2d, animals, respectively. Chimeric mice were then infected with Lm, and disease outcome was monitored. BALB/c T cell chimeric mice, BALB/c ATXBM hosts given naive C57BL/6.C-H-2d T cells, resolved their infections as indicated by reductions in both lesion size and parasite numbers. Furthermore, the mice developed typical Th1 (interferon[IFN]-gamma hiinterleukin[IL]-4lo) cytokine patterns. In contrast, both sham chimeric, BALB/c ATXBM hosts given naive BALB/c T cells, and control irradiated euthymic mice succumbed to infection, producing Th2 profiles (IFN-gamma loIL-4hiIL-10hi). C57BL/6 T cell chimeras, C57BL/6 ATXBM hosts given naive BALB.B6-H-2b T cells, resolved their infections as did C57BL/6 sham chimeras and euthymic controls. Interestingly, whereas C57BL/6 control animals produced Th1 cytokines, chimeric animals progressed from Th0 (IFN-gamma hiIL-4hiIL-10hi) to Th2 (IFN-gamma loIL-4hiIL-10hi) cytokine profiles as cure ensued. Both reconstitution and chimeric status of all mice were confirmed by flow cytometry. In addition, T cell receptor V beta usage of Lm-specific blasts was determined. In all cases, V beta use was multiclonal, involving primarily V beta 2, 4, 6, 8.1, 8.2, 8.3, 10, and 14, with relative V beta frequencies differing between H-2b and H-2d animals. Most importantly, however, these differences did not segregate between cure and noncure outcomes. These findings indicate that: (a) genetic traits determining cure in Lm infection can direct disease outcome from both T cell and non-T cell compartments; (b) the presence of the curing genotype in only one compartment is sufficient to confer cure; (c) curing genotype T cells autonomously assume a Th1 cytokine profile-mediating cure; (d) noncuring genotype T cells can mediate cure in a curing environment, despite the onset of Th2 cytokine production; and lastly, (e) antigen specificity of responding T cells, as assessed by V beta T cell receptor diversity, is not a critical determinant of disease outcome.
The T cell response of HLA-DR transgenic mice to human myelin basic protein and other antigens in the presence and absence of human CD4.
Authors: Altmann D M , Douek D C , Frater A J , Hetherington C M , Inoko H , Elliott J I
Published: June 2004

Analysis of HLA class II transgenic mice has progressed in recent years from analysis of single chain HLA class II transgenes with expression of mixed mouse/human heterodimers to double transgenic mice expressing normal human heterodimers. Previous studies have used either HLA transgenic mice in which there is a species-matched interaction with CD4 or mice which lack this interaction. Since both systems are reported to generate HLA-restricted responses, the matter of the requirement for species-matched CD4 remains unclear. We have generated triple transgenic mice expressing three human transgenes, DRA, DRB, and CD4, and compared HLA-restricted responses to peptide between human-CD4+ (Hu-CD4+) and Hu-CD4- littermates. We saw no difference between Hu-CD4+ and Hu-CD4- groups, supporting the notion that for some responses at least the requirement for species-matched CD4 may not be absolute. Evidence for positive selection of mouse T cell receptors in HLA-DR transgenic mice came both from the acquisition of new, HLA-restricted responses to various peptides and from an increased frequency of T cells using the TCR V beta 4 gene segment. An important goal with respect to the analysis of function in HLA transgenic mice is the clarification of mechanisms which underpin the recognition of self-antigens in human autoimmune disease. As a first step towards 'humanized' disease models in HLA transgenic mice, we analyzed the responses of HLA-DR transgenic mice to the human MPB 139-154 peptide which has been implicated as an epitope recognized by T cells of multiple sclerosis patients. We obtained T cell responses to this epitope in transgenic mice but not in nontransgenic controls. This study suggests that HLA transgenic mice will be valuable in the analysis of HLA-restricted T cell epitopes implicated in human disease and possibly in the design of new disease models.
Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells.
Authors: Baum L G , Pang M , Perillo N L , Wu T , Delegeane A , Uittenbogaart C H , Fukuda M , Seilhamer J J
Published: June 2004

Thymic epithelial cells play a crucial role in the selection of developing thymocytes. Thymocyte-epithelial cell interactions involve a number of adhesion molecules, including members of the integrin and immunoglobulin superfamilies. We found that human thymic epithelial cells synthesize an endogenous lectin, galectin-1, which binds to oligosaccharide ligands on the surface of thymocytes and T lymphoblastoid cells. Binding of T lymphoblastoid cells to thymic epithelial cells was inhibited by antibody to galectin-1 on the epithelial cells, and by two antibodies, T305 and 2B11, that recognize carbohydrate epitopes on the T cell surface glycoproteins CD43 and CD45, respectively. T lymphoblastoid cells and thymocytes bound recombinant galectin-1, as demonstrated by flow cytometric analysis, and lectin binding was completely inhibited in the presence of lactose. The degree of galectin-1 binding to thymocytes correlated with the maturation stage of the cells, as immature thymocytes bound more galectin-1 than did mature thymocytes. Preferential binding of galectin-1 to immature thymocytes may result from regulated expression of preferred oligosaccharide ligands on those cells, since we found that the epitope recognized by the T305 antibody, the core 2 O-glycan structure on CD43, was expressed on cortical, but not medullary cells. The level of expression of the UDP-GlcNAc:Gal beta 1,3GalNAc-R beta 1, 6GlcNAc transferase (core 2 beta 1, 6 GlcNAc transferase, or C2GnT), which creates the core 2 O-glycan structure, correlated with the glycosylation change between cortical and medullary cells. Expression of mRNA encoding the C2GnT was high in subcapsular and cortical thymocytes and low in medullary thymocytes, as demonstrated by in situ hybridization. These results suggest that galectin-1 participates in thymocyte-thymic epithelial cell interactions, and that this interaction may be regulated by expression of relevant oligosaccharide ligands on the thymocyte cell surface.
Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively.
Authors: Takagi M , Hara T , Ichihara M , Takatsu K , Miyajima A
Published: June 2004

The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony-stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice that express the mouse IL-5R alpha constitutively by phosphoglycerate kinase promoter. The transgenic mouse expressed IL-5R alpha ubiquitously, and the bone marrow cells formed various types of colonies, including multi-lineage colonies, in response to IL-5. IL-5 also supported formation of both multi-lineage and blast cell colonies from dormant progenitors of the 5-fluorouracil-treated transgenic mice. The cells composing the blast cell colony gave rise to many colonies including multi-lineage colonies when they were replated in secondary culture containing either Il-5 or IL-3. There was no significant difference in replating efficiency or in types of secondary colonies between IL-5- and IL-3-stimulated cultures. Conversely, the cells from the IL-3-induced blast cell colonies of the transgenic mice proliferated in response to either IL-3 or IL-5. Thus, the development of the progenitors can be equally supported by either IL-5 or IL-3, suggesting that intracellular signals from the IL-3R can be replaced by those from IL-5. These results strongly suggest that the lineage specificity of IL-5 is mainly due to the restricted expression of IL-5R alpha.
Mechanism of interleukin 12-mediated toxicities during experimental viral infections: role of tumor necrosis factor and glucocorticoids.
Authors: Orange J S , Salazar-Mather T P , Opal S M , Spencer R L , Miller A H , McEwen B S , Biron C A
Published: June 2004

Interleukin 12 (IL-12) doses in excess of 100 ng/d have been shown to induce profound immunotoxicities in mice infected with lymphocytic choriomeningitis virus (LCMV). These immunotoxicities are characterized by almost complete inhibition of virus-induced CD8+ T cell expansion and CTL activation, and up to 2 log increases in viral replication. They are accompanied by induction of serum tumor necrosis factor (TNF). The studies presented here were undertaken to characterize mechanisms for the IL-12-induced toxicities and to examine expression and function of TNF in this context. Several physiological changes were induced in IL-12-treated uninfected and dramatically elevated in IL-12-treated virus-infected mice. IL-12 induced (a) decreases in body weights, > 10% in uninfected and > 20% in LCMV-infected mice; (b) elevation of circulating glucocorticoid levels to > 10 micrograms/dl in uninfected and > 20 micrograms/dl in infected mice; and (c) decreases in thymic mass, > 30% in uninfected and up to 95% in infected mice. These changes are known to be associated with circulating TNF. Northern blot and in situ hybridization analyses demonstrated that IL-12 induced TNF-alpha expression and that LCMV infection synergized with IL-12 for induction of this factor. Antibodies neutralizing TNF reversed all of the IL-12-induced toxicities in LCMV-infected mice including the immunotoxicities against CD8+ T cells and anti-viral defenses. The TNF-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and CD8+ T cell subsets isolated from LCMV-infected mice were more sensitive to TNF-mediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. Experiments with the glucocorticoid type II receptor antagonist, RU486, demonstrated that endogenous glucocorticoids were secondary intermediaries in IL-12-induced thymic atrophy. Studies in IL-2-deficient mice showed that the synergism was dependent upon endogenous IL-2. The results delineate a unique mechanism of TNF-mediated toxicity. In addition, they have significant implications concerning potential detrimental consequences of in vivo TNF induction and of IL-12 administration for protective anti-viral responses.
T cell receptor-mediated signaling events in CD4+CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection.
Authors: Wang C R , Hashimoto K , Kubo S , Yokochi T , Kubo M , Suzuki M , Suzuki K , Tada T , Nakayama T
Published: June 2004

The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocked the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression were inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FK506 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FK506. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes.
CD8 beta chain influences CD8 alpha chain-associated Lck kinase activity.
Authors: Irie H Y , Ravichandran K S , Burakoff S J
Published: June 2004

The CD8 molecule plays an important role in the differentiation of CD8+ T cells in the thymus and in their normal function in the periphery. CD8 exists on the cell surface in two forms, the alpha alpha homodimer and the alpha beta heterodimer. Recent studies indicate an important role for the CD8 beta chain in thymic development of CD8+ T cells and suggest that signaling via CD8 alpha beta may be distinct from CD8 alpha alpha. To better understand these differences, we introduced the CD8 beta gene into a T cell hybridoma which only expressed the CD8 alpha alpha homodimer. In the parent hybridoma, cross-linking of the CD8 alpha chain led to minimal enhancement of CD8-associated Lck tyrosine kinase activity. In the CD8 beta+ transfectants, several observations suggested that CD8 beta modifies CD8 alpha-associated Lck tyrosine kinase activity: (a) in in vitro kinase assays, antibody-mediated crosslinking of CD8 alone, or CD8 cross-linking with the TCR, resulted in 10-fold greater activation of Lck kinase activity, compared to cells expressing CD8 alpha alpha alone; (b) in vivo, markedly enhanced tyrosine phosphorylation of several intracellular proteins was observed upon CD8 cross-linking with the TCR in CD8 alpha beta-expressing cells, compared to cells expressing CD8 alpha alpha alone; and (c) Lck association with CD8 alpha was stabilized by the coexpression of CD8 beta. Thus, the differential Lck kinase activation and tyrosine phosphorylation seen with CD8 alpha alpha vs. CD8 alpha beta may reflect the unique signaling capabilities of the CD8 beta molecule. These differences in signaling may, in part, account for the diminished ability to generate CD8 single positive thymocytes in mice bearing a homozygous disruption of the CD8 beta gene.
The antigen-presenting activities of Ia+ dendritic cells shift dynamically from lung to lymph node after an airway challenge with soluble antigen.
Authors: Xia W , Pinto C E , Kradin R L
Published: June 2004

Dendritic cells (DC) are widely distributed in the lung where they are distinguished by their morphology and class II major histocompatibility complex (Ia) antigen expression. Although a role for DC as pulmonary antigen-presenting cell (APC) has been suggested, little is currently known concerning how these cells respond to inhaled antigens in vivo. Hen-egg lysozyme (HEL) was injected intratracheally into Lewis rats; DC were subsequently purified from the lung and regional lymph nodes (LN) at intervals of up to 14 d and examined for their ability to stimulate the proliferation of HEL-immune T cells in vitro in the absence of added HEL. Pulmonary DC displayed APC activities at 3 h and for up to 7 d after the injection of antigen. Dendritic cells in the draining hilar LN showed APC activities that appeared at 24 h, peaked at day 3, and then diminished progressively. After the primary sensitization, HEL-immune T cells were detected in hilar LN but not in the lung. A second airway challenge with HEL at day 14 yielded an antigen-specific pulmonary immune response, characterized histologically by the accumulation of mononuclear cells around lung venules. We conclude that APC activities shift from lung to lymph node during the response to inhaled antigen.
Poliovirus-specific CD4+ Th1 clones with both cytotoxic and helper activity mediate protective humoral immunity against a lethal poliovirus infection in transgenic mice expressing the human poliovirus receptor.
Authors: Mahon B P , Katrak K , Nomoto A , Macadam A J , Minor P D , Mills K H
Published: June 2004

The current understanding of the function of CD4+ T helper (Th) cells in immunity to infectious diseases is that Th1 cells, which secrete interleukin (IL)-2 and interferon-gamma, induce cellular immune responses, whereas Th2 cells, which secrete IL-4, IL-5, IL-6, and IL-10, provide helper function for humoral immunity. We have used a panel of poliovirus-specific murine CD4+ T cell clones and mice transgenic for the human poliovirus receptor to evaluate the role of Th cell subpopulations in protective immunity to poliovirus. The majority of T cell clones, as well as polyclonal T cells generated from mice infected or immunized with poliovirus, secreted IL-2 and interferon-gamma, but not IL-4, IL-5, or IL-10, a profile typical of Th1 cells. The Th1 clones displayed major histocompatibility complex class II-restricted cytotoxic T lymphocyte activity against specific poliovirus peptide-pulsed target cells, but also provided help for antipoliovirus neutralizing antibody production. To examine the mechanism of immunity in vivo, we have used poliovirus receptor-transgenic mice on a BALB/c (H-2d) background. These animals developed a poliomyelitis-like disease when challenged intravenously with a virulent wild-type strain of poliovirus, but not with an attenuated vaccine strain. Furthermore, mice immunized with the vaccine strain were protected against a subsequent challenge with wild-type virus. Using an adoptive transfer technique, we demonstrated that it was possible to confer protection with primed B cells in the presence of polyclonal poliovirus-specific T cells, but not when transgenic mice received either B cells or T cells alone. Furthermore, protection was observed when mice received primed B cells in the presence of a VP4-specific Th1 clone. The findings demonstrate that Th1 cells can mediate a protective immune response against poliovirus infection in vivo through helper activity for humoral immunity and that CD4+ T cells, specific for the internal poliovirus capsid protein, VP4, can provide effective help for a protective antibody response directed against surface capsid proteins.
Infection of gamma/delta T lymphocytes by human herpesvirus 6: transcriptional induction of CD4 and susceptibility to HIV infection.
Authors: Lusso P , Garzino-Demo A , Crowley R W , Malnati M S
Published: June 2004

Human herpesvirus 6 (HHV-6), a T-lymphotropic human herpesvirus, is a potentially immunosuppressive agent that has been suggested to play a role as a cofactor in the natural history of human immunodeficiency virus (HIV) infection. We studied the interactions between HHV-6 and gamma/delta T lymphocytes, a subset of T cells involved in the protective immune response against specific microorganisms. Polyclonal gamma/delta T cell populations, purified from the peripheral blood of healthy adults and activated in vitro with phytohemagglutinin, were exposed to HHV-6, strain GS (subgroup A), at the approximate multiplicity of infection (MOI) of 1. Signs of virus replication were detected as early as 72 h after infection, as documented by immunofluorescence, electron microscopy, and transmission of extracellular virus. Progression of the infection was associated with the appearance of typical cytomorphological changes and, eventually, massive cell death. In contrast, no signs of infection or cytopathic effects were detected after exposure of gamma/delta T lymphocytes to HHV-7, a CD4+ T-lymphotropic virus closely related to HHV-6. Polyclonal gamma/delta T cells displayed cytolytic activity against both autologous and heterologous target cells infected with HHV-6 and maintained this activity for at least 72 h after infection with HHV-6, despite the high MOI used. As previously documented in mature CD8+ alpha/beta T cells and natural killer cells, HHV-6 infection induced gamma/delta T lymphocytes to express de novo CD4 messenger RNA and protein, as detected by reverse transcriptase-polymerase chain reaction and fluorocytometry, respectively. Whereas purified CD4- gamma/delta T cell populations were per se refractory to HIV infection, they became susceptible to productive infection by HIV-1, strain IIIB, after induction of CD4 expression by HHV-6. These results demonstrate that gamma/delta T cells can be directly targeted and killed by a herpesvirus and may have implications for the potential role of HHV-6 in AIDS.
The Duffy antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Duffy negative individuals who lack the erythrocyte receptor.
Authors: Peiper S C , Wang Z X , Neote K , Martin A W , Showell H J , Conklyn M J , Ogborne K , Hadley T J , Lu Z H , Hesselgesser J , Horuk R
Published: June 2004

The Duffy antigen/receptor for chemokines (DARC), first identified on erythrocytes, functions not only as a promiscuous chemokine receptor but also as a receptor for the malarial parasite, Plasmodium vivax. The recent finding that DARC is ubiquitously expressed by endothelial cells lining postcapillary venules provides a possible insight into the function of this receptor because this anatomic site is an active interface for leukocyte trafficking. However, the biological significance of DARC is questionable since it has not yet been determined whether individuals lacking the expression of this protein on their erythrocytes (Duffy negative individuals), who are apparently immunologically normal, express the receptor on endothelial cells. However, we report here that DARC is indeed expressed in endothelial cells lining postcapillary venules and splenic sinusoids in individuals who lack the erythrocyte receptor. These findings are based on immunohistochemical, biochemical, and molecular biological analysis of tissues from Duffy negative individuals. We also present data showing that, in contrast to erythrocyte DARC, cells transfected with DARC internalize radiolabeled ligand. We conclude that the DARC may play a critical role in mediating the effects of proinflammatory chemokines on the interactions between leukocyte and endothelial cells since the molecular pathology of the Duffy negative genotype maintains expression on the latter cell type.
Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells.
Authors: Xie K , Huang S , Dong Z , Juang S H , Gutman M , Xie Q W , Nathan C , Fidler I J
Published: June 2004

Previous studies from our laboratory demonstrated an inverse relationship between the expression level of inducible nitric oxide synthase (iNOS) and the metastatic potential of murine K-1735 melanoma cells. The purpose of this study was to provide direct evidence that the expression of iNOS suppresses metastatic potential of melanoma cells. Highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containing 3 mM NG-methyl-L-arginine (NMA). Positive transfectants were identified by Southern and Northern blot analyses and homogeneous staining with a specific anti-iNOS monoclonal antibody. Semiconfluent cultures of C4.P (parental), C4.Neo.3 (control transfection), C4.S2.3 (inactive iNOS), and C4.L8.5 (functional iNOS) were harvested, and viable cells were injected intravenously into syngeneic C3H/HeN mice and allogeneic BALB/c nude mice. C4.P, C4.Neo.3, and C4.S2.3 cells were highly metastatic whereas C4.L8.5 cells were not metastatic. Experiments with [125I]dUrd-labeled tumor cells demonstrated that the initial arrest in the lung microvasculature did not differ among the lines, but that C4.L8.5 cells died by 48-72 h after injection. Enhanced survival of all K-1735 C4 cells (including C4.L8.5) was found in mice given twice daily injections of 20 mg NMA. The C4.L8.5 cells produced slow growing subcutaneous tumors in nude mice, whereas the other three lines produced fast growing tumors. In vitro studies confirmed that in the absence of NMA the expression of iNOS in C4.L8.5 cells induced apoptosis. Collectively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis.
Mutational analysis and an alternatively spliced product of B7 defines its CD28/CTLA4-binding site on immunoglobulin C-like domain.
Authors: Guo Y , Wu Y , Zhao M , Kong X P , Liu Y
Published: June 2004

Costimulatory molecules B7 and B7-2 interact with T cell surface receptors CD28/CTLA4 and deliver a costimulatory signal essential for T cell growth. However, the structure basis of this interaction is not known. B7 and B7-2 are members of immunoglobulin (Ig) superfamily and their extracellular portion consists of an IgV- and IgC-like domain. Here we report that a naturally occurring, alternatively spliced form of B7 reveals that exon 3-encoded IgC domain is essential for CD28/CTLA4 binding. Mutational analysis of B7 demonstrates a critical role of several amino acids around loops between strands B and C and D and E, for binding CTLA4/CD28. These amino acids are clustered to form a single binding site centered at 201Y. A comparison of the effects of mutations on the binding of CD28 and CTLA4 reveals that CD28 and CTLA4 binds to the same site on B7. These results have important implications on the role of CTLA4 and CD28 in T cell costimulation. The structure of the CD28/CTLA4-binding site also provides valuable information for immune intervention targeted at the B7/B7-2-CD28/CTLA4 interactions.
The FLT3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors.
Authors: Jacobsen S E , Okkenhaug C , Myklebust J , Veiby O P , Lyman S D
Published: June 2004

The recently cloned murine flt3 ligand (FL) was studied for its ability to stimulate the growth of primitive (Lin-Sca-1+) and more committed (Lin-Sca-1-) murine bone marrow progenitor cells, alone and in combination with other hematopoietic growth factors (HGFs). Whereas FL was a weak proliferative stimulator alone, it potently synergized with a number of other HGFs, including all four colony-stimulating factor (CSF), interleukin (IL) 6, IL-11, IL-12, and stem cell factor (SCF), to promote the colony formation of Lin-Sca-1+, but not Lin-Sca-1- or erythroid progenitor cells. The synergistic activity of FL was concentration dependent, with maximum stimulation occurring at 250 ng/ml, and was observed when cells were plated at a concentration of one cell per culture, suggesting that its effects are directly mediated. 2 wk of treatment with FL in combination with IL-3 or SCF resulted in the production of a high proportion of mature myeloid cells (granulocytes and macrophages), whereas the combination of FL with G-CSF, IL-11, or IL-12 resulted predominantly in the formation of cells with an immature blast cell appearance. Accordingly, FL in combination with G-CSF or IL-11 expanded the number of progenitors more than 40-fold after 2 wk incubation. Thus, FL emerges as a potent synergistic HGF, that in combination with numerous other HGFs, can directly stimulate the proliferation, myeloid differentiation, and expansion of primitive hematopoietic progenitor cells.