bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute rejection of allogeneic hemopoietic progenitors by genetically resistant mice
Authors: Shimamura Kazuo , Kaminsky Steven G. , Nakamura Ichiro
Published: March 2007

Natural resistance to hemopoietic allograft results in functional elimination of the graft from the host within 24 to 48 h. The resistance is specific and is directed to the cell surface target structures of an unidentified nature. These determinants are controlled by the major histocompatibility complex‐linked hemopoietic histocompatibility (Hh) loci and expressed by cells of the hemopoietic system. In this study, the susceptibility of various hemopoietic progenitors, as well as the nature and the kinetics of the early rejection process, were examined by directly following the functional survival of the grafted progenitors by periodic sampling. In both F1 hybrid and inbred allogeneic hosts, multipotential progenitors for granulocyte‐erythrocyte‐monocyte‐megakaryocyte lineages, bipotential progenitors for granulocyte‐monocyte lineages and unipotential progenitors of erythrocyte, granulocyte and monocyte were susceptible to elimination. Therefore, within the limited range of comparisons, neither the lineage, nor the degree of commitment or differentiation determines the susceptibility of hemopoietic progenitors to resistance. Functional elimination of the grafted progenitors from the recipient spleen was irreversible upon cultivation in semisolid medium. The frequency of seeding in the spleen at 3 h was comparable in resistant and susceptible hosts elimination commenced shortly afterwards and by 24 h after transplantation < 10% of clonogenic progenitors remained functional. Activation of natural killer cells strengthened pre‐existing resistance, but did not convert genetically susceptible mice to resistant. Therefore, the resistance is dependent on an effector mechanism with predetermined specificity.
Loss of interleukin 2 dependence in cloned interleukin 2‐dependent rat T lymphocyte × BW5147 hybridomas is not associated with segregation of a specific pair of rat chromosomes
Authors: Somoza Chamorro , Fernández‐Ruiz Elena , Jotterand‐Bellomo Martine , Sanz Eva , Nabholz Markus , Silva Augusto
Published: March 2007

A fusion between the mouse AKR thymoma BW5147 and a culture of homogeneously OX8+ (CD8) rat T lymphoblasts yield interleukin (IL) 2‐dependent T cell hybridomas when selected in HAT medium supplemented with IL 2‐containing supernatants of concanavalin A‐activated cells and dexamethasone. IL 2‐independent variants can be selected from cloned IL 2‐dependent hybrids in the absence of conditioned medium. Karyotype analysis was used to test a previously proposed hypothesis according to which IL 2‐independent variants arise through loss of a specific cytotoxic T lymphocyte (rat) chromosome carrying a gene responsible for IL 2 dependence. Comparison of karyotypes of several independently derived hybrids with those of their IL 2‐independent variants showed that the hybrids contain at least one homologue of all rat chromosomes, and that no pair of rat chromosomes is consistently absent in the IL 2‐independent variants.
CD15 antibodies increase neutrophil adhesion to endothelium by an LFA‐1‐dependent mechanism
Authors: Forsyth Kevin D. , Simpson Anna C. , Levinsky Roland J.
Published: March 2007

Anti‐neutrophil antibodies (CD15) bind to a simple sugar (lactose‐N‐fucopentaose III, LNF III) known to be present on the chains of the adhesion molecules on neutrophils. We have demonstrated that pre‐incubation of neutrophils with a CD15 antibody increases neutrophil adherence to endothelium by a neutrophil‐dependent mechanism. This augmented adhesion can be inhibited by antibodies directed against the α and β chain of the leukocyte function‐associated antigen 1 (LFA‐1) molecule. There is also some increase in surface LFA‐1 expression on neutrophils after CD15 incubation, suggesting that CD15 antibodies increase neutrophil adhesion by an LFA‐1‐dependent mechanism. The increase in LFA‐1 expression after CD15 incubation occurs in the presence of a protein synthesis inhibitor. As LFA‐1 is not stored intracellularly, the increased adherence of the neutrophils, and increased LFA‐1 expression on their surface, suggests the possibility that the CD15 antibodies are binding to the LNF III antigen present on the α and β chains of LFA‐1, producing their effect by activating the molecule, perhaps by exposing new antigen sites by a stearic effect.
Human T cells and interleukin 4 inhibit the release of interleukin 1 induced by lipopolysaccharide in serumfree cultures of autologous monocytes
Authors: Weiss Laurence , Haeffner‐Cavaillon Nicole , Laude Maryline , Cavaillon Jean‐Marc , Kazatchkine Michel D.
Published: March 2007

Stimulation with lipopolysaccharide (LPS) of unfractionated peripheral blood mononuclear cells in serum‐free cultures resulted in the release of lower amounts of interleukin (IL) 1 as compared with those induced in cultures of purified monocytes. In addition, a dose‐dependent decrease in IL 1 production was observed when purified autologous T cells were added to cultures of LPS‐stimulated monocytes. The inhibitory effect of T cells on IL 1 production was reproduced by adding human recombinant IL4 to monocyte cultures. IL4 optimally inhibited the generation of cell‐associated IL 1 and the extracellular release of IL 1 in monocyte cultures performed in the presence of low doses of LPS. IL 4 inhibited the generation of IL 1 activity and that of IL 1α and IL 1β antigens indicating that IL4 interfered with IL 1 transcription and/or processing rather than induced the synthesis of an IL1 inhibitor. IL2 and IL3 enhanced IL1 production by LPS‐stimulated cells whereas granulocyte‐monocyte colony‐stimulating factor had no effect. IL4 inhibited the production and the release of tumor necrosis factor α activity by monocytes stimulated with LPS. Thus, T cells and IL4 may down‐regulate the production of monokines which induce their proliferation and exert a suppressive effect on the generation of potent proinflammatory mediators.
Monokine production by microglial cell clones
Authors: Righi Marco , Mori Lucia , Libero Gennaro De , Sironi Marina , Biondi Andrea , Mantovani Alberto , Donini Suzanne Denis , Ricciardi‐Castagnoli Paola
Published: March 2007

Cytokines have been suggested to act as intermediates between the immune and the central nervous system, but little is known about the type of cells synthesizing them in the brain. We have immortalized with oncogenic retroviruses primary brain cell cultures from mouse embryos and have generated clones of microglial cells that have been characterized. Three of the clones studied produce interleukin 1 (IL 1), IL 6 and tumor necrosis factor‐α as assessed by biological assays and by Northern blot analysis. Our data raise the question on the role of these cytokines in the brain and suggest that early resident microglial cells might play an important role in developmental processes and in the adult brain.
MALA‐2, mouse homologue of human adhesion molecule ICAM‐1 (CD54)
Authors: Prieto Jacqueline , Takei Fumio , Gendelman Rita , Christenson Birger , Biberfeld Peter , Patarroyo Manuel
Published: March 2007

In humans, lymphocyte adhesion to cells is mediated by the protein heterodimer CD11a/CD18 (Leu‐CAMa, LFA‐1) and its ligand CD54 (ICAM‐1). Although the murine CD11a/CD18 is well characterized, the mouse homologue of human ICAM‐1 has not been identified. In the present study a rat monoclonal antibody to the murine lymphocyte activation antigen MALA‐2 was found to inhibit in a dose‐dependent manner the phorbol ester‐enhanced aggregation of mouse lymphoblasts, an adhesionspecific assay, and hence to define an adhesion molecule. By immunofluorescence flow cytometry the antigen expression was low on spleen cells but it largely increased after stimulation with mitogens. The antigen was expressed by some, but not all, lymphoid cell lines, and myelomonocytic and mastocytoma cells were also positive. In frozen tissue sections MALA‐2 was mainly detected on germinal center B cells, dendritic cells, macrophages and vascular endothelium, including high endothelial venules. Cell surface labeling followed by immunoprecipitation and gel electrophoresis indicated that the antigen is a sialoglycoprotein which has a relative molecular mass of 95 kDa and displays a faster electrophoretic mobility under nonreducing conditions. The function, cellular distribution and molecular properties of MALA‐2 are indistinguishable from those of human ICAM‐1.
Transcriptional regulation of immunoglobulin expression in a chronic lymphocytic leukemia cell line
Authors: Mårtensson Inga‐Lill , Nilsson Kenneth , Leanderson Tomas
Published: March 2007

A cell line derived from a B‐type chronic lymphocytic leukemia, Corinna II, was found to down‐regulate its steady‐state level of IgM mRNA after treatment with phorbol 12‐myristate 13‐acetate while the proliferative capacity of the cell line was unaffected. No changes in the splicing pattern of the IgM transcript could be observed after treatment. The down‐regulatory effect on IgM RNA expression was found to be inhibited by cycloheximide, suggesting that functional protein synthesis was needed for the effect. Transfection experiments showed that the down‐regulatory effect of phorbol myristate acetate was exerted at the level of transcriptional initiation. The DNA element mediating the down‐regulatory effect was found to be present within 140 bp 5' of the mRNA cap site in an immunoglobulin promoter.
Inactivation of rabbit immunoglobulin λ chain variable region genes by the insertion of short interspersed elements of the C family
Authors: Hayzer David J. , Jaton Jean‐Claude
Published: March 2007

Two rabbit germ‐line genes encoding immunoglobulin λ light chain V regions were cloned from a rabbit genomic liver DNA library and characterized. One, Vλ1, is separated by at least 8 kb from any other Vλ gene. The second, Vλ4, forms part of a three‐gene cluster with two functional Vλ genes recently reported. Both Vλ1 and Vλ4 have structural features rendering them pseudogenes. The coding regions have frame‐shift mutations which would yield defective protein products; both genes are also interrupted by the insertions of short, interspersed repetitive elements of the C family. In the Vλ1 gene, the 369‐bp insert is located upstream of the gene between the putative TATA box and the leader exon, whereas in gene Vλ4, the 360‐bp insert interrupts the FR2 at codon 48c. In addition, the sequence of the complement‐determining region 3 of gene Vλ1 is very similar to the mouse DSP2.6 sequence.
Clonal evidence for the induction of NKH1 on activated human thymocytes
Authors: Caligiuri Michael A. , Murray Christine , Levine Herbert , Longtine Janina A. , Ritz Jerome
Published: March 2007

Freshly isolated human thymocytes lack the NKH1 antigen and the ability to lyse target cells without major histocompatibility complex restriction. Short‐term culture of human thymocytes in interleukin (IL) 2 results in the generation of non‐major histocompatibility complex‐restricted effector cells, all of which express NKH1. The mechanism by which these cells appear in culture has yet to be elucidated. In the present studies, we developed thymocyte clones and performed a molecular analysis of T cell receptor gene rearrangements to demonstrate that the expression of NKH1 antigen is induced on the surface of NKH1‐ thymocytes in the presence of IL2. In addition, we were able to show that the NKH1+ fraction consistently displayed an increased proliferative response to similar concentrations of IL 2 when compared to NKH1‐ cells, for both clonal and polyclonal populations of thymocytes. Taken together, these studies demonstrate that the initial appearance of the NKH1 antigen following thymocyte culture in the presence of IL2 results from the induction of NKH1 expression on NKH1‐ thymocytes, while the subsequent predominance of this cell type also results from an enhanced proliferative response to IL 2 which coincides with NKH1 expression.
Restricted usage of VH and VX genes by murine monoclonal antibodies against 3‐fucosyllactosamine
Authors: Kimura Hirohisa , Buescher E. Stephen , Ball Edward D. , Marcus Donald M.
Published: March 2007

We are studying the structure and regulation of murine antibodies against the 3‐fucosyllactosamine antigenic determinant. Analysis of the sequences of seven BALB/ c IgM,χ; monoclonal antibodies (mAb), obtained from four fusions, indicates that these antibodies exhibit restriction in their usage of VH and VL genes. Based on a combination of mRNA sequences and Southern filter hybridization data, all seven light chains are encoded by Vχ;24B and Jχ;1 gene segments. Complete mRNA sequences of the heavy chains revealed that all seven mAb are encoded by VH441, six antibodies are encoded by JH4 and one uses a Jn3 gene segment. The VH441 gene segment and all seven mAb contain a potential glycosylation site at Asn 58 in complementarity‐determining region (CDR)2. In contrast to the similarity of the VH regions, the heavy chain CDR3 segments exhibit considerable heterogeneity. They are encoded by three D segments, they vary in length from 7‐9 amino acids and display differences in their deduced amino acid sequences. The VH441 gene segment also encodes antibodies against four other carbohydrate antigens, levan, galactan, dextran and galactosyl globoside. The use of a single gene segment to encode antibodies against five different antigens suggests that the domain encoded by VH441 might be particularly well adapted for forming sites that bind carbohydrate determinants. Glycosylation of CDR2 might contribute to the unique properties of this VH domain.
Non‐random migration of cd4 and cd8 t cells: changes in the cd4: cd8 ratio and interleukin 2 responsiveness of efferent lymph cells following in vivo antigen challenge
Authors: Bujdoso Raymond , Young Patricia , Hopkins John , Allen Deborah , Mcconnell Ian
Published: March 2007

In this investigation we have examined some of the cellular and molecular changes in efferent lymph that drains from an antigen‐stimulated peripheral lymph node. Resting efferent lymph is characterized by a higher percentage of CD4+ cells and consequently, a higher CD4/CD8 ratio than peripheral blood. Following antigen stimulation of a cannulated peripheral lymph node in antigen‐primed sheep, the percentage of CD4+ cells in efferent lymph increases above the resting level during days 1, 2 and 3 post antigen stimulation. This is followed on days, 3, 4, and 5 after antigen stimulation by an increase in the percentage of CD8+ cells above the resting level which occurs as the percentage of CD4+ cells returns to the resting level. These changes cause the CD4/CD8 ratio to first increase above the resting value during the CD4 phase and then decrease below the resting value during the CD8 phase. During the CD4 phase a lymphokine activity is present in cell‐free lymph fluid. Lymph fluid collected at this time supports the proliferation of activated T cells. Supernatants generated from efferent cells collected at a similar time and cultured in vivo for 24 h without any further stimulation are capable of releasing this material. During the CD8 phase cells expressing functional interleukin(IL) 2 receptors appear in lymph fluid. The data suggests a sequential exit of T cell subsets from an antigen‐stimulated lymph node and that the appearance of IL 2‐like activity and IL 2‐responsive cells in efferent lymph fluid are temporally distinct events.
Bioactive human recombinant tumor necrosis factor‐α: an unstable dimer?
Authors: Petersen Claus Munck , Nykjær Anders , Christiansen Brian S. , Heickendorff Lene , Mogensen SøRen C. , Møller Bjarne
Published: March 2007

As determined by native polyacrylamide gel electrophoresis (PAGE) and gel chromatography the molecular mass of native tumor necrosis factor (TNF)‐α was approximately 35 kDa. When incubated at low concentrations (< 1 nM) 125I‐labeled TNF‐α and unlabeled TNF‐α rapidly multimerized or dissociated into monomers and bioactivity decreased. Sodium dodecyl sulfate (SDS)‐PAGE analysis of cross‐linked 125I‐labeled TNF‐α demonstrated bands of multi‐ and trimeric TNF‐α in addition to dominating bands of dimers and monomers. Tri‐, di‐ and monomeric TNF‐α were recovered from SDS‐PAGE gels and allowed to renature. Of the original receptor‐binding activity, 10%–15% was obtained with cross‐linked TNF‐α dimers, whereas none was recovered from preparations of trimeric TNF‐α. Multimeric and monomeric TNF‐α exhibited little or no binding activity, and cell‐bound, cross‐linked TNF‐α which was dissociated from cellular binding sites was mainly dimeric. 125I‐labeled TNF‐α bound to lymphokine‐activated killer (LAK) cells and binding kinetics were much similar (Kd approximately 100 pM) to those reported in other normal cell types. The number of receptors per LAK cell was approximately 4 × 103. Cross‐linking of TNF‐α to binding sites in U‐937 and LAK cells yielded a receptor‐ligand complex of about 80/90 kDa. At 37 °C, 125I‐labeled TNF‐α was rapidly internalized and degraded in L‐929, U‐937 and LAK cells. Degradation of ligand and recycling of receptors were blocked in the presence of methylamine. Methylamine significantly inhibited TNF‐α‐mediated cytolysis of L‐929 cells and caused a quantitatively corresponding reduction in cellular TNF‐α uptake, indicating that L‐929 lysis was mediated by receptors.
Effects of lipopolysaccharide on macrophages analyzed with anti‐lipid A monoclonal antibodies and polymyxin B
Authors: Lasfargues AgnÈS , Tahri‐Jouti Mohamed‐Ali , Pedron Thierry , Girard Robert , Chaby Richard
Published: March 2007

Six monoclonal antibodies (mAb) to the lipid A region of bacterial lipopolysaccharide (LPS), obtained from mice immunized with lipid A‐coated Bordetella pertussis cells (mAb 3.E8, 2.21, 2.37, 2.41) or with lipid A covalently coupled to keyhole limpet hemocyanin (mAb R1 and R7), were examined for their potential to inhibit in vitro activities of LPS on macrophages. mAb R7 was inactive in vitro, but the five other mAb inhibited efficiently some in vitro activities of LPS. mAb R1, 2.21 and 3.E8 reduced the LPS‐induced secretion of tumor necrosis factor (TNF) and interleukin 1 (IL 1) by macrophages, but did not modify the binding of LPS to macrophages. On the other hand, mAb 2.37 and 2.41 reduced LPS binding to macrophages and subsequent IL 1 secretion, but did not modify TNF production. This is in agreement with our previous finding that IL 1 and TNF productions can be selectively triggered by synthetic analogs of lipid A substructures (Lasfargues and Chaby, Cell. Immunol. 1988. 115: 165). The pattern of in vitro inhibition of LPS activities (LPS binding to macrophages and production of TNF and membrane IL 1) by polymyxin B was different from those of the two groups of anti‐lipid A mAb mentioned above. These observations suggest the presence on lipid A of four functionally distinct substructures.
Anti‐inflammatory activity of human IgA antibodies and their Fabα fragments: inhibition of IgG‐mediated complement activation
Authors: Russell Michael W. , Reinholdt Jesper , Kilian Mogens
Published: March 2007

The interaction of human IgA antibodies with the classical pathway of complement activation was investigated in a homologous human system, by means of two IgA1 and three IgG1 myeloma proteins having antibody activity against a defined antigen, staphylococcal α‐toxin. In a solid‐phase antigendependent C3b‐binding ELISA system, the monoclonal IgG antibodies were previously shown to activate the classical complement pathway synergistically, resembling polyclonal IgG antibodies, whereas IgA antibodies were unable to activate complement by either pathway. In the present study, IgA antibodies were found to inhibit significantly the activation of complement initiated by antigenbound polyclonal or mixed monoclonal IgG antibodies, in relation to the amount of IgA antibodies applied and bound to antigen. IgA1 myeloma proteins devoid of antigen‐binding activity were without effect. Inhibition was independent of the ability of the IgA antibodies to compete against the IgG antibodies in binding to antigen, and was demonstrable with physiological concentrations of antibodies. Similar results were obtained with polyclonal serum IgA having antigen‐binding activity. However, the binding of C1q to antigen‐complexed IgG was inhibited only by a monoclonal IgA antibody that could compete against one of the three monoclonal IgG antibodies that bound C1q synergistically. This observation implied that at least two mechanisms were involved in the inhibition of C3b fixation. Fabα fragments of monoclonal IgA antibodies, obtained by cleavage with IgA1 protease from Haemophilus influenzae type b, were found to have a similar inhibitory effect on C3b fixation to the intact IgA1 antibodies. This observation supports the hypothesis that IgA1 proteases contribute to the invasive pathogenicity of certain mucosal bacteria, by cleaving secretory IgA1 antibodies to antigen‐binding Fabα fragments, which are not only defective in mucosal defense properties, but which also protect the organisms from other immune effector systems, such as complement activation.
Contrasting effects of glucocorticoids on the capacity of T cells to produce the growth factors interleukin 2 and interleukin 4
Authors: Daynes Raymond A. , Araneo Barbara A.
Published: March 2007

The molecular mechanisms which govern the biosynthesis and secretion of the various T cell‐derived lymphokines are poorly understood at this time, in spite of their tremendous importance to the control of the mammalian immune system. Here we provide compelling evidence that production of the murine T cell growth factors interleukin (IL)2 and IL 4 are differentially regulated by glucocorticoid (GCS) hormones. Under conditions where IL 2 production is reduced by GCS hormones, IL 4 production is increased. In vivo, this effect on T cell production of growth factors is manifest at low GCS concentrations that are well within physiologic ranges. In vitro, splenocytes isolated from antigen‐stimulated donors, as well as antigen‐specific cloned T cell lines, undergo alterations in their capacity to secrete T cell growth factors when stimulated with antigens in the presence of GCS. Responses normally dominated by IL 2 are dramatically shifted to a condition where IL 4 represents the major species of T cell growth factor produced. Similar changes in the pattern of T cell growth factor production are observed following short pulses with low‐dose GCS in vitro, and the steroid‐induced depression in IL 2 production can be reversed and/or inhibited by treatment with the potent steroid antagonist RU486. Our results imply that GCS hormones, presumably through their capacity to activate a specified family of ligand‐dependent transcriptional regulatory proteins (steroid hormone receptors), function to control the pattern of lymphokines produced by activated T cells. Steroid‐mediated regulation of lymphokine gene expression could serve to dictate the types of immune effector mechanisms which can be initiated subsequent to antigen exposure.
Cholera toxin modulates the T cell antigen receptor/CD3 complex but not the CD2 molecule and inhibits signaling via both receptor structures in the human T cell lymphoma Jurkat
Authors: Sommermeyer Henning , Schwinzer Reinhard , Kaever Volkhard , Wessel Klaus , Schmidt Reinhold E. , Behl Berthold , Wonigeit Kurt , Szamel Marta , Resch Klaus
Published: March 2007

The human T cell lymphoma Jurkat can be activated by stimuli directed either against the T cell antigen receptor‐CD3 antigen complex (TcR/CD3) or the CD2 molecule. Stimulation of cells via the TcR/CD3‐complex or via the CD2 molecule increases inositol phosphates and cytoplasmic free calcium. Pretreatment of Jurkat cells with cholera toxin leads to a decrease of TcR/CD3 expression on the surface of the cells, while the expression of CD2 is unaffected. In contrast to this distinct effect on the receptor expression, signaling via both pathways is inhibited by cholera toxin. The most convincing explanation for the cholera toxin‐mediated inhibition of signaling is that cholera toxin interrupts the signaling pathways at a point where both, stimulation via TcR/CD3 and via CD2, use the same route. The earliest common point of the two signaling pathways, at least in the Jurkat cell line, seems to be the CD3 complex because after its down‐regulation (and functional inactivation) both pathways of activation are interrupted.
Synergistic action of calcium ionophore A23187 and protein kinase C activator bryostatin 1 on human B cell activation and proliferation
Authors: Drexler Hans G. , Gignac Suzanne M. , Pettit George R. , Hoffbrand A. Victor
Published: March 2007

In this study we have examined the immunostimulatory effects of the macrocyclic lactone bryostatin 1 on various aspects of B cell activation and proliferation using human tonsillar B cells. Bryostatin 1 is an activator of protein kinase C (PKC) and its properties were compared to those of the classical PKC activator phorbol 12‐myristate 13‐acetate (PMA), a phorbol ester. Time‐course kinetics and dose‐response curves of RNA and DNA synthesis induced by bryostatin 1 or PMA were comparable, albeit the phorbol ester was significantly more potent. The responses triggered by both bryostatin 1 and PMA could be blocked by the PKC inhibitor H7. Bryostatin 1 and PMA mediated similar effects with regard to the activation parameters, increase in cell size, expression of activation‐associated antigens and hyperexpression of major histocompatibility complex class II antigens. Addition of the calcium ionophore A23187 to bryostatin 1‐treated cultures resulted in synergistically enhanced activation and proliferation responses, and this potentiation by A23187 could be inhibited by cyclosporin A. Bryostatin 1 antagonized the effects of PMA‐triggered stimulation in a time‐ and dose‐dependent manner. The basis for this modulation of PMA‐induced effects and the reason for the difference in the abilities of the two agents to stimulate B cells is unclear; possibly, bryostatin 1 and PMA activate different isoforms of PKC and elicit different signals on intracellular biochemical pathways.

Bryostatin 1 lacks the tumor‐promoting activity of PMA and is a potent anti‐neoplastic substance. These features together with its immunomodulatory properties qualify bryostatin 1 as a candidate for in vivo use as a biological response modifier.
Differential activity of interleukin 1α and interleukin 1β in the stimulation of the immune response in vivo
Authors: Boraschi Diana , Villa Luigi , Volpini Gianfranco , Bossù Paola , Censini Stefano , Ghiara Paolo , Scapigliat Giuseppe , Nencioni Luciano , Bartalini Marcella , Matteucci Giacomo , Cioli Franca , Carnasciali Marzia , Olmastroni Enza , Mengozzi Manuela , Ghezzi Pietro , Tagliabue Aldo
Published: March 2007

The biological activities of human recombinant interleukin (IL) 1α and IL 1β were compared in different biological systems. The two IL 1 forms were equally active in vitro in inducing proliferation of murine thymocytes and of the murine T helper clone D10.G4.1, and in triggering release of prostaglandin E2 from human skin fibroblasts. In vivo, IL 1α and IL 1β were similarly pyrogenic both in rabbits and mice, and could equally increase the circulating levels of the acute phase protein serum amyloid A in mice. However, only IL 1β showed immunostimulatory activity in vivo, as it could enhance the number of specific antibody‐producing cells in the spleen of mice immunized with either a T‐dependent or a T‐independent antigen. Although devoid of immunostimulatory activity, IL 1α could efficiently compete immunostimulation induced by IL 1β, suggesting an effective interaction with the IL 1 receptor. Thus, IL 1β appears to have an important role in the positive regulation of immune responses, while IL 1α may act as down‐regulator of the IL 1β effect.
Purification and characterization of a 26‐kDa competitive inhibitor of interleukin 1
Authors: Mazzei Gonzalo J. , Seckinger Philippe L. , Dayer Jean‐Michel , Shaw Alan R.
Published: March 2007

The urine of patients with fever above 39 °C contains an inhibitor of interleukin 1. Herein we describe the purification of the interleukin 1 inhibitor by ion‐exchange chromatography, hydrophobic chromatography, gel filtration and negative immunosorption. The purified protein has a molecular weight of 26000; its size is reduced to 24000 upon treatment with endoglycosidase F. The IL 1 inhibitor is a competitive inhibitor and acts by binding to the IL 1 receptor. The inhibitor recognizes the murine and human IL 1 receptor with similar affinity. The purified IL 1 inhibitor has a specific activity of 3 × 107 to 4.5 × 107 units/mg as tested on the human astrocytoma (U‐373) cell proliferation bioassay and murine EL4.6.1 cell IL 1 receptor‐binding assay.
Lymphokines and bacteria, that induce tumoricidal activity, trigger a different secretory response in macrophages
Authors: Keller Robert , Keist Ruth , Frei Karl
Published: March 2007

The abilities of various macrophage‐activating agents to trigger tumoricidal activity and/or the secretion of prostaglandin E2 (PGE2), interleukin 6 (IL 6) and transforming growth factor β (TGFβ) in bone marrow‐derived mononuclear phagocytes (BMMΦ) in vitro were comparatively assessed. Induction of tumoricidal activity by lymphokines, that is only short‐lived, was not associated with enhanced secretion of these activities by BMMΦ; in contrast, incubation with heat‐killed facultative intracellular bacteria resulted in persisting tumoricidal activity and in marked enhancement of the secretion of IL 6 and PGE2, but not of TGFβ activity. These findings support the concept that the pattern of the secretory response induced in macrophages by lymphokines differs from that triggered by bacteria and that the rapid decay of lymphokine‐induced tumoricidal activity is not due to autocrine macrophage deactivation mediated by one of these agents alone.
CD4+CD8+ thymocytes are susceptible to DNA fragmentation induced by phorbol ester, calcium ionophore and anti‐CD3 antibody
Authors: Tadakuma Takushi , Kizaki Harutoshi , Odaka Chikako , Kubota Ryo , Ishimura Yuzuru , Yagita Hideo , Okumura Ko
Published: March 2007

Stimulation of murine thymocytes with phorbol ester or calcium ionophore for 18–24 h resulted in 70%–80% fragmentation of DNA into 180–200‐bp multiples, followed by cell death. Experiments with fractionated subpopulations by panning or flow cytometry revealed that DNA fragmentation was selectively observed in CD4+CD8+ cells and in a portion of CD4−CD8+ cells. To investigate whether DNA cleavage is also inducible via antigen‐specific receptors, thymocytes were incubated in wells precoated with anti‐CD3 antibody. An approximately 20% increase of DNA fragmentation was constantly observed when unseparated thymocytes were stimulated with anti‐CD3 antibody. In this anti‐CD3‐induced DNA degradation, CD4+CD8+ cells are probably the target cells, since (a) fetal thymocytes at day 18 of gestation were found vulnerable to anti‐CD3‐induced DNA cleavage and (b) flow cytometry analysis of viable cells recovered after cultivation in the anti‐CD3‐coated wells revealed that CD4+CD8+ cells were preferentially decreased. Further experiments with purified CD4+CD8+ cells, however, could not define a clear‐cut increase of DNA fragmentation when isolated CD4+CD8+ cells were stimulated with anti‐CD3 antibody. Addition of interleukin (IL) 1, IL 2, IL 3, IL 4 or interferon‐γ to the CD4+CD8+ cell cultures failed to yield a DNA cleavage similar to that of unseparated thymocytes.
T cell receptor gene rearrangements of T lymphocytes infiltrating the liver in chronic active hepatitis B and primary biliary cirrhosis (PBC): Oligoclonality of PBC‐derived T cell clones
Authors: Moebius Ulrich , Manns Michael , Hess Georg , Kober Gabriela , zum Büschenfelde Karl‐Herrmann Meyer , Meuer Stefan C.
Published: March 2007

Immunological events are involved in the pathophysiology of chronic active hepatitis as indicated from the accumulation of T lymphocytes at the site of tissue damage. We generated T cell clones from liver biopsies of 3 patients with chronic active hepatitis B and 2 patients with primary biliary cirrhosis. These T cell clones (n = 84) were analyzed by means of T cell receptor (TcR) β gene rearrangements to determine whether the infiltrate consists of a polyclonal or oligoclonal T cell population. The vast majority (62 of 64) of T cell clones from three different patients with chronic active hepatitis B showed no identical rearrangements of the TcR β chain genes. In marked contrast, in both patients with primary biliary cirrhosis, T cell clones established were of limited diversity. Thus 5 out of 10 and 2 out of 10 T cell clones from one patient and 3 out of 9 and 2 out of 9 T cell clones from the second patient, respectively, showed identical TcR β gene rearrangements. These data suggest that a clonal dominance is characteristic for local T cell responses in autoimmune liver disease such as primary biliary cirrhosis whereas in virus‐induced chronic active hepatitis T cell activation occurs polyclonally.
Natural killer cells carry the germ‐line configuration of the T cell receptor δ chain gene and heterogeneously express six distinct δ transcripts
Authors: Carè Alessandra , Pelicci Pier G. , Meccia Ettore , Fagioli Marta , Testa Ugo , Ciccone Ermanno , Moretta Alessandro , Moretta Lorenzo , Peschle Cesare
Published: March 2007

Investigations on T cell receptor δ chain (TcR δ) gene expression in freshly isolated normal large granular lymphocytes, natural killer (NK) cell clones and NK bulk cultures revealed six Tδ transcripts (3.5, 3.1, 2.2, 2.0, 1.5 and 1.3 kb) which varied in number and relative abundance in the different samples. None of the six known Tδ variable regions was expressed and the Tδ locus was retained in its germ‐line configuration. The origin and significance of these NK‐associated Tδ transcripts are discussed.
Detection of intracellular expression and secretion of interferon‐γ at the single‐cell level after activation of human T cells with tetanus toxoid in vitro
Authors: Kabilan Lalitha , Andersson Gudrun , Lolli Francesco , Ekre Hans‐peter , Olsson Tomas , Troye‐blomberg Marita
Published: March 2007

Activation of Tcells results in intracellular expression and secretion of cytokines such as interferon (IFN)‐γ. Here we have used three different assays for determination of IFN‐γ in tetanus toxoid‐ or mitogen‐activated human T cell cultures. Two of these assays [intracytoplasmic immunofluorescence and enzyme‐linked immuno spot assay (ELISPOT)] determined the expression and secretion of IFN‐γ at the single‐cell level while the third assay enzyme‐linked immunosorbent assay (ELISA) measured IFN‐γ secreted into the culture supernatant. Comparison of all three tests revealed a good correlation between the ELISPOT assay and the ELISA, whereas expression of intracellular IFN‐γ showed a qualitative but not a quantitative correlation with the latter. Both the immunospot assay and the immunofluorescence may be used to detect approximate numbers of specific T cells even when present at low frequencies. With the use of the immunospot assay antigen‐specific T cells could be detected even in the absence of detectable IFN‐γ in the culture supernatants. However, the ELISA assay should be more convenient for screening large clinical material.
Tumor necrosis factor‐α in combination with interferon‐γ, but not with interleukin 4 activates murine macrophages for elimination of Leishmania major amastigotes
Authors: Bogdan Christian , Moll Heidrun , Solbach Werner , Röllinghoff Martin
Published: March 2007

We have previously shown that during an infection with Leishmania major, susceptible BALB/c mice, as opposed to mice of a resistant strain (C57BL/6), are primed by lipopolysaccharide for the production of high levels of tumor necrosis factor‐α (TNF‐α) which is known to be a potent macrophage (MΦ) stimulator in other parasitic diseases. In the present study we investigated whether TNF‐α activates MΦ for killling of L. major parasites. In the absence of interferon‐γ (IFN‐γ) or lipopolysaccharide, TNF‐α (0.025—25000 U/ml) failed to activate peritoneal exudate MΦ from BALB/c micefor killling of L. major amastigotes. In the presence of suboptimal doses of IFN‐γ (5 or 10 U/ml), however, TNF‐α mediated a rapid elimination of intracellular parasites, which was highly significant compared to IFN‐γ alone. The combination of TNF with interleukin 4, in contrast, was inactive in this respect and allowed survival of intracellular parasites. From these data we conclude that thepresence of IFN‐γ is curcial for TNF‐α‐mediated killing of L. major parasites by MΦ. Disease progression in susceptible mice thereforeseems to be a consequence of a deficiency of IFN‐γ and a predominance of interleukin 4 rather than the result of an excess amount of TNF‐α.
Inhibition of interleukin 4 receptor expression on human lymphoid cells by cyclosporin
Authors: Foxwell Brian M. J. , Woerly Gaetane , Ryffel Bernhard
Published: March 2007

The effect of the immunosuppressant cyclosporin (CsA) on the expression of interleukin (IL) 4 membrane receptors on human peripheral blood mononuclear cells (PBMC) was investigated after cell activation by anti‐CD3 antibody, IL 2 or IL 4. Previous studies with 125I‐labeled IL 4 identified on resting lymphocytes a trimolecular complex consisting of a 65/70‐kDa doublet and a 110‐kDa protein with ∼︁ 300 high‐affinity binding sites (Kd 100 pM) and ∼︁ 9000 low‐affinity binding sites (Kd 30 nM). Upon cell activation by anti‐CD3 antibody both low‐ and high‐affinity binding sites increased about threefold concomitant with upregulation of all the cross‐linked proteins. CsA inhibited anti‐CD3 antibody‐induced up‐regulation of IL 4 receptor (IL 4R)‐associated proteins as well as the expression of high‐affinity binding sites. However, up‐regulation of IL 4R by its own ligand or IL 2 and the growth‐promoting effect of IL 4 on activated, IL 4R+ T cells were CsA resistant. Since CsA inhibits the synthesis of IL 4, exogenous IL 4 was added to the cultures and it partially reversed the inhibitory effect of CsA on cell proliferation as well as on IL 4R expression. It is concluded that the inhibitory effect of CsA on IL 4R expression may contribute to the immunosuppressive effect of the drug.
Dissociation of unidirectional influx of external Ca2+ and release frominternal stores in activated human T lymphocytes*
Authors: Gelfand Erwin W. , Cheung Roy K.
Published: March 2007

Addition of lectin or antibody to the T cell receptor complex of human T cells results in a rapid increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This response is biphasic and results from contributions of Ca2+ from internal stores, uptake of Ca2+ across the plasma membrane and possibly a decrease in Ca2+ efflux. These responses have been linked through the activity of inositol 1,4,5‐trisphosphate in releasing Ca2+ from internal stores and potentially mediating Ca2+ uptakeacross the plasma membrane. Following addition of phytohemagglutinin or anti‐CD3 antibody to resting T cells or Jurkat cells, we have been able to dissociate the [Ca2+]i responses by loading cells with the Ca2+ chelator 1,2‐bis(Oaminophenoxy)ethaneN,N,N',N'‐tetraacetate (BAPTA). In BAPTA‐loaded T cells, we have shown that Ca2+ mobilized from intracellular stores following activation is effectively buffered, while stimulated Ca2+ uptake and associated changes in [Ca2+]i were relatively unaffected. In this report, we show that the sustained increase in [Ca2+]i is due to increased unidirectional influx of external Ca2+ without changes in efflux and that it is the entry of extracellular Ca2+ which is sensitive to the transmembrane potential.
Definition of the human immunoglobulin variable lambda (IGLV) gene subgroups
Authors: Chuchana Paul , Blancher Antoine , Brockly Frédérique , Alexandre Daniel , Lefranc Gérard , Lefranc Marie‐paule
Published: March 2007

Comparison of 60 human immunoglobulin variable lambda (IGLV) sequences allowed us to define seven subgroups designated VλI to VλVII. We demonstrate that all λ proteins sequenced so far fall into the subgroups I, II, III and VI, and that the λ regions previously assigned to subgroups IV and V belong, in fact, to subgroups III and II, respectively. Four sequences not belonging to any of the subgroups I, II, III and VI define the new subgroups IV, V and VII. Interestingly, these subgroups show a higher homology to rabbit or mouse Vλ genes than to the other human Vλ subgroups. By comparison of the proteins either with the sequences deduced from the germ‐line genes or with the consensus sequences, therate of amino acid changes due to somatic mutations or allelic variations was evaluated in several λ proteins. Framework and complementarity‐determining regions of the human IGLV genes and proteins were delineated. Alignment of the λ sequences shows that functional V‐J rearrangement occurs, with or without deletion of nucleotides encoding one or two amino acids at the 3′ end of the V gene. Diversity of the third complementarity‐determining region is due to somatic mutations and to flexibleV‐J junction, but there is no evidence of N‐diversity in the human λlocus.
T cell activation by a bispecific anti‐CD3/anti‐major histocompatibility complex class I antibody
Authors: Roosnek Eddy , Tunnacliffe Alan , Lanzavecchia Antonio
Published: March 2007

A bispecific anti‐CD3/anti‐major histocompatibility complex (MHC) class I antibody (Ab) that is able to activate human T cells in the absence of a second signal has been used to compare activation by this Ab to signaling by anti‐CD3 Ab orby antigen (Ag). We have studied early, intermediate and late events that occur after triggering of a tetanus toxoid‐specific T cell clone. While bivalent anti‐CD3 Ab induce transient (<20 min) intracellular Ca2+ concentration increases, the bispecific Ab, like antigen‐presenting cells plus Ag, produces sustained (> 2 h) elevated intracellular Ca2+ concentration. In addition, while the anti‐CD3 Ab only induces low levels of c‐myc mRNA lasting less than 3 h, the bispecific Ab induces high levels which are maintained for at least 8 h after triggering. Late events, such as interleukin 2 receptor expression and proliferation, occurring more than 20 h after activation, were seen only when T cells were stimulated by bispecific Ab or by antigen‐presenting cell and Ag. Cells activated by linking T cell receptor(TcR)/CD3 to MHC class I antigens could not be activated by cross‐linking the MHC and TcR/CD3 structures separately. Therefore, we propose that the bispecific Ab functions by modifying the membrane dynamics of the TcR/CD3 complex, rather than by the generation of a second signal through class I antigens.
Characterization of the CD3 γ and δ invariant subunits of the sheep T cell antigen receptor
Authors: Hein Wayne R. , Tunnacliffe Alan
Published: March 2007

The CD3 γ and δ chains of the sheep T cell antigen receptor have been characterized by cDNA cloning. In comparison to human and mouse protein sequences, short oligopeptide stretches have been deleted from both the γ and δ chains. However, apart from further variability in the number and position of potential N‐linked glycosylation sites, the general structure of the CD3 γ and δ proteins is markedly conserved. Particularly remarkable are two motifs consisting of 8 and 32 amino acids, respectively, which locate in the C‐terminal half of each protein and which have been precisely conserved in both the CD3 γ and δ chains of all three species. These motifs probably contribute structural properties essential for the function of the CD3/T cell receptor complex. mRNA of the sheep CD3 γ (1.5 and 1.2 kb) and CD3 δ (1.5 kb) chains are transcribed from closely linked, single‐copy genes, suggesting that common mechanisms for the regulation of CD3 genes are also preserved between species.
Comparison of diversity and function of house dust mite‐specific T lymphocyte clones from atopic and non‐atopic donors
Authors: Wierenga Eddy A. , Snoek Mieke , Bos Jan D. , Jansen Henk M. , Kapsenberg Martien L.
Published: March 2007

Panels of CD4+CD8− T lymphocyte clones (TLC), specific for house dust mite Dermatophagoides pteronyssinus (Dp) proteins, were generated from the peripheral blood of an atopic Dp‐allergic donor (AD), suffering from severe atopic dermatitis, and a histocompatible non‐atopic donor (NAD). We studied the diversity of TLC within these two panels in search for the possible occurrence of dominant clone types with properties that might be characteristic for the atopic or non‐atopic state. TLC with specificities for at least four different Dp proteins were found within the panel from AD “L” and for at least three different Dp proteins within the panel from NAD “K”. In addition, both panels showed a considerable but comparable restriction diversity within HLA‐DR. Despite the diversity within the panels, all Dp‐specific TLC from AD were found to produce IL 4, after HLA class II‐restricted Dp‐specific stimulation, whereas the TLC from NAD produced no or only minimal amounts of this lymphokine. Only supernatants from stimulated AD TLC could induce IgE secretion by B cells from NAD. Conclusively, these observations do not give evidence for the occurrence of an abnormal Dp‐specific T cell repertoire in AD, but rather suggest aberrant secretion of the IgE‐inducing lymphokine IL 4 by CD4+ Dp‐specific T cells from AD.
Enhanced immunogenicity of recombinant peptide fusions containing multiple copies of a heterologous T helper epitope
Authors: Löwenadler Björn , Svennerholm Ann‐Mari , Gidlund Magnus , Holmgren Erik , Krook Katarina , Svanholm Cecilia , Ulff Suzanne , Josephson Staffan
Published: March 2007

We have examined the immune response against the nonimmunogenic heatstable enterotoxin (STa) of enterotoxigenic Escherichia coli using recombinant fusion proteins containing the STa‐peptide linked to an IgG‐binding analogue of protein A and varying numbers of the T helper epitope 323–339 from ovalbumin (ova). By immunization of inbred strains of mice with a series of STa fusion proteins, containing up to four copies of ova tandemly multiplied, we demonstrated that the anti‐STa antibody response is controlled by ova‐specific T helper cells in a genetically restricted manner. In the responding mouse strains (2 out of 3 tested), the level of antibody production was increased by addition of multiple ova epitopes, the anti‐STa response being considerably higher to fusion proteins containing four than one or two ova epitopes.
IgE‐dependent antigen focusing by human B lymphocytes is mediated by the low‐affinity receptor for IgE
Authors: Pirron Ulrich , Schlunck Tilman , Prinz Jörg C. , Rieber E. Peter
Published: March 2007

In this study we investigated the role of the low‐affinity receptor for IgE (FcϵRII, CD23) on Epstein‐Barr virus (EBV)‐transformed human B cells in the uptake and presentation to T cells of antigen after complexing with IgE. Cloned EBV‐transformed B cells were incubated for 5 h with (4‐hydroxy‐3‐iodo‐5‐nitrophenyl)acetyl (NIP)‐haptenized tetanus toxoid (NIP‐TT) or NIP‐TT complexed with a chimeric human IgE/mouse anti‐NIP monoclonal antibody (IgE × NIP‐TT) and then contacted for 2 min with autologous cloned TT‐specific T cells. Intracellular Ca2+ mobilization in T cells was determined as an early indicator of T cell activation. The antigen‐presenting capacity of B cells was significantly increased by complexing the antigen with IgE. This effect could be selectively reversed in a dose‐dependent manner by blocking the FcϵRII with an anti‐CD23 monoclonal antibody. The IgE‐mediated increased capacity for presenting antigen became particularly evident when B cells were incubated with NIP‐TT or IgE × NIP‐TT for only 1 h at 4 °C, washed and then cultivated for 6 h at 37 °C allowing uptake and processing of the antigen. These results indicate a new role of the FcϵRII/CD23 molecules in the uptake of antigen by APC which might be of importance in the maintenance of an ongoing immune response against allergens.