bluuebunny/crossref_metadata_2025 huggingface dataset:
L1 adhesion molecule on mouse leukocytes: regulation and involvement in endothelial cell binding
Authors: Hubbe Marcus , Kowitz Aiga , Schirrmacher Volker , Schachner Melitta , Altevogt Peter
Published: March 2007

LI is a cell surface glycoprotein of the immunoglobulin superfamily which was initially shown to mediate adhesion between neural cells. Recently we have reported that LI is expressed by bone marrow cells and the majority of mature lymphocytes (Kowitz et al., Eur. J. Immunol. 1992. 22: 1199‐1205). To analyze the function of LI on leukocytes we studied its regulation following cell activation. In vitro activation of B lymphocytes with lipopolysaccharide or T lymphocytes with phorbol 12‐myristate 13‐acetate/Ca2+ ionophore, concanavalin A or anti‐CD3 monoclonal antibody as well as in vivo activation of Vβ8+ T cells with staphylococcal enterotoxin B (SEB) revealed a down‐regulation of LI within 48 h. A rapid loss of LI expression was seen when mouse neutrophils were activated with PMA alone. This rapid loss paralleled the shedding of L‐selectin.

We also studied a possible role of LI in the binding of leukocytes to endothelial cells. ESb‐MP lymphoma cells with a high expression of L1 (L1hi) could bind to bend3 endothelioma cells without prior activation with inflammatory cytokines. The interaction was inhibited by anti‐L1 antibodies. In contrast, ESb‐MP cells with low LI expression (L110) were only marginally bound. Latex beads coated with affinity‐isolated LI antigen were also able to bind to the endothelioma cells in a specific fashion. The binding of ESb‐MP lymphoma cells required Ca2+ and Mg2+ ions and was sensitive to cold temperature. Since the endothelioma cells did not express LI the binding mechanism studied here is distinct from the established L1‐L1 homotypic interaction. It is possible that the novel L1‐mediated adhesion pathway involves an unidentified ligand and could play a role in leukocyte migration.
Fine restriction analysis and inhibition of antigen recognition in HLA‐DQ‐restricted T cells by major histocompatibility complex blockers and T cell receptor antagonists
Authors: Serra Horacio M. , Crimi Claire , Sette Alessandro , Celis Esteban
Published: March 2007

The role of polymorphic residues of the β chain of human histocompatibility leukocyte antigen‐DQw5/w6 in antigen presentation to a hepatitis B surface antigen‐specific Tcell clone was studied. The results obtained demonstrate that the residue situated at position 57 of the β chain (a valine) is critical for presentation of antigen by antigen‐presenting cells to the DQ‐restricted T cell clone. Experiments were also done to study the feasibility of peptide blocking of antigen recognition by DQ‐restricted T cells. The results indicate that peptides known to associate with DQ molecules are capable of blocking the presentation of antigen to the DQ‐restricted Tcell clone, presumably by competing with antigen for binding to major histocompatibility complex (MHC) molecules. Moreover, truncations of the stimulatory antigenic peptide resulted in the production of Tcell receptor antagonists, which inhibited the response of the T cells to antigen at 10–100‐fold lower concentrations than conventional MHC blockers. The role of DQ‐restricted Tcell responses and peptide blocking approaches in autoimmunity are discussed.
TH2 activated cells prevent experimental autoimmune uveoretinitis, a TH1‐dependent autoimmune disease
Authors: Saoudi Abdelhadi , Kuhn Joelle , Huygen Kris , de Kozak Yvonne , Velu Thierry , Goldman Michel , Druet Philippe , Bellon Blanche
Published: March 2007

Mercuric chloride (HgCl2) injections protect (Lewis x Brown‐Norway) F1 (F1) rats against experimental autoimmune uveoretinitis (EAU) induced by immunization with the retinal S antigen (S‐Ag); in contrast HgCl2‐injected F1 rats develop EAU following transfer of lymph node (LN) cells from rats immunized with S‐Ag alone. In the present study we demonstrate that the ability of LN cells from rats protected against EAU to transfer the disease into naive F1 rats was considerably reduced. These LN cells neither produced interleukin (IL)‐2 nor (interferon (IFN)‐γ but exhibited mRNA for IL‐4. In contrast, LN cells from diseased rats easily transferred EAU into naive F1 rats, produced significant IL‐2 and IFN‐γ levels but barely exhibited mRNA for IL‐4. Furthermore protected rats predominantly produced IgG2b anti‐S‐Ag antibodies, while diseased rats produced IgG2b anti‐S‐Ag antibodies and the increase in expression of MHC class II molecules on B cells was higher in protected rats than in diseased rats. These data suggest that (1) to exert a protective effect, HgCl2 must act at an early stage of differentiation of precursors of S‐Ag specific T cells, and (2) this effect is related to the preferential activation of TH2 cells to the detriment of uveitogenic TH1 cells. Finally, these results indicate that activation of TH2 cells protect from a TH1‐dependent autoimmune disease.
Interleukin‐5 expressed by a recombinant virus vector enhances specific mucosal IgA responses in vivo
Authors: Ramsay Alistair J. , Kohonen‐Corish Maija
Published: March 2007

Several in vitro studies have shown that murine interleukin‐5 (mIL‐5) enhances IgA production by activated mucosal B cells. To date, however, there is no evidence that this factor significantly up‐regulates mucosal IgA responses in vivo. Here, we show that expression of the gene for mIL‐5 in a recombinant vaccinia virus vector markedly increases IgA responses to co‐expressed heterologous antigen in the lungs of mice given intranasal inocula of the virus. The elevated local IgA responses to vectors expressing mIL‐5 peaked at a fourfold higher level than those elicited by control virus at 14 days after infection and were sustained for at least 4 weeks. Increased IgA responses were abrogated in mice treated with monoclonal antibody against mIL‐5 and were not detected in systemic lymphoid tissue. No enhancement of specific IgG levels was found either locally or systemically. Our results indicate that mIL‐5 selectively enhances the development of mucosal IgA responses in vivo and suggest that expression of this factor in mucosal vaccine vectors may stimulate local immune reactivity.
β2‐Adrenergic stimulation causes detachment of natural killer cells from cultured endothelium
Authors: Benschop Robert J. , Oostveen Frank G. , Heijnen Cobi J. , Ballieux Rudy E.
Published: March 2007

Physical exercise, mental stress, or infusion of β‐adrenergic agonists result in an increase in the number of natural killer (NK) cells in the peripheral circulation. In view of the specific migration pattern of NK cells in vivo, it has been suggested that these cells may be released from the marginating pool in blood vessels. In the present report, the in vitro effect of catecholamines on the adhesion of NK cells to unstimulated human endothelial cells (EC) was characterized. Peripheral blood mononuclear cells were allowed to adhere to monolayers of EC, after which the adherent lymphocyte fraction was analyzed phenotypically by flow cytometry. NK cells were found to adhere preferentially to EC, a process that was reversed by the addition of various adrenergic agonists. Catecholamines selectively affected adhesion of NK cells and had no effect on T cell adhesion to EC, as was determined by the use of purified cell populations. Detachment of NK cells from EC could be achieved by short incubations (5 min) with epinephrine (EPI) and was concentration‐dependent, with an ED50 of 2 × 10 −10M. Using a panel of α and β‐adrenergic agonists and antagonists, we show that the detachment of NK cells is mediated via β2‐adrenergic receptors. In line with the lower affinity for β2‐adrenergic receptors, norepinephrine was less effective than EPI in inducing detachment of NK cells from EC. Direct activation of adenylate‐cyclase with forskolin gave similar results as observed with EPI, indicating that signaling through cAMP is necessary to induce detachment of NK cells from EC. The results of the present study lend support to the hypothesis that catecholamines, via β2‐adrenergic receptors, can induce recruitment of NK cells from the marginating pool to the circulating pool, by changing the adhesive interactions between NK cells and EC.
Expressed human immunoglobulin ϰ genes and their hypermutation
Authors: Klein Ralph , Jaenichen Rita , Zachau Hans G.
Published: March 2007

The question of which germ‐line Vϰ genes are expressed was studied by sequencing 70 different cDNA clones from a human spleen library and one clone from a fetal liver library. The sequences were compared to a data base containing all germ‐line Vϰ gene and pseudogene sequences. In addition, 51 rearranged genomic Vϰ genes, 170 cDNA and 74 × proteins from the literature were assigned to specific germ‐line Vϰ genes and included in the comparisons. Not all the known, potentially functional Vϰ genes were found to be expressed, while some genes with minor defects are. The total number of expressed genes is smaller than expected: so far 21 germ‐line genes and 5 pairs of duplicated identical genes are known to be transcribed. The corresponding numbers for rearranged genomic VH genes and x proteins are 17 plus 4 and 7 plus 7, respectively. A second aim of the study was to find out whether the expressed repertoire contains derivatives of germ‐line Vϰ genes still missing in our data base; no evidence for the existence of such genes was found.

Several cDNA clones contained additional nucleotides between the Vϰ and Jϰ gene segments, which may be germ‐line derived, inserted by terminal deoxynu‐cleotidyl transferase or introduced by other mechanisms. Somatic gene conversion seems not to play a major role in creating the human ϰ gene diversity. Various aspects of the hypermutation of ϰ genes are discussed and the formation of block mutations, i.e. the alterations of two or more adjacent nucleotides is stressed as a remarkable feature of the process.
Chemical reactivity of an HLA‐B27 thiol group
Authors: Whelan Michael Anthony , Archer James Ronald
Published: March 2007

Techniques have been developed to measure the reactivity of free thiols in the HLA class I antigen‐binding cleft. HLA‐B27, which sequencing predicts has a free cysteine at position 67, reacts rapidly with the positively charged thiol reagent monobromotrimethyl‐ammoniobimane bromide (qBBr) to give products which are identifiable by isoelectric focusing. HLA‐B38, B39, B64 and B65, all of which have a similar Cys 67, react less strongly. Several other class I molecules, notably HLA‐C antigens, are reactive in this system, and it may be capable of recognizing subtypes such as A*0207 which also carry free cysteine. The accessibility of thiol to qBBr depends both on the chemistry of the class I molecule and other factors in the cell. Two human cell lines which are known to carry identical B27 genes but do not present the same peptides, differ considerably in the accessibility of their B27 thiol. Evidence from mouse cells transfected with mutant B27 genes suggests that a unique lysine at position 70 in the wild‐type molecule increases reactivity to thiol‐reactive metabolites. The failure of B27 to give a complete reaction with qBBr in our model systems suggests that it can exist in more than one chemical form. This may leave the molecule susceptible to oxidation, causing errors in T cell recognition and an exaggerated inflammatory response.
A role for antigen‐presenting cells and bacterial superantigens in reversal of human T lymphocyte anergy
Authors: Burns James , Littlefield Kimberly
Published: March 2007

The induction of anergy in T lymphocytes generates T cells incapable of proliferation in response to a conventional antigenic stimulus. To investigate the induction and maintenance of anergy in human T cells, we used T cell‐T cell presentation of myelin basic protein (MBP) or MBP synthetic peptides to induce anergy in vitro. Although anergic T cells responded normally to interleukin‐2 (IL‐2), these T cells did not produce IL‐2 or IL‐4 when peripheral blood mononuclear cells presented MBP or MBP peptides. Proliferation of anergic T cells was reduced by greater than 95% compared to nonanergic, control T cells. However, when autologous B cell lines were used to present MBP, anergy was partially reversed with a proliferation response about 50% of nonanergic levels. Bacterial superantigens also partially restored proliferation in anergic T cells following presentation by either B cell lines or macrophage isolated from peripheral blood mononuclear cells. Anergic, MBP‐reactive T cells fully retained antigen‐specific cytolytic activity against both B cell and T cell targets presenting MBP. These results suggest that T cell proliferative anergy may be reversible with both the type of antigen‐presenting cell and superantigens potentially contributing to the initiation or maintenance of an autoimmune response.
Deficient antigen processing of a protein quaternary structure can be overcome by receptor‐mediated uptake
Authors: Rouas‐Freiss Nathalie , Housseau Franck , Bidart Jean‐Michel , Bonnerot Christian , Amigorena Sebastien , Guillet Jean‐Gérard , Bellet Dominique
Published: March 2007

Human chorionic gonadotropin (hCG) is a dimer of non‐covalently associated alpha (hCG‐α) and beta (hCG‐β) subunits. This molecule was used to study whether receptor‐mediated uptake influences the presentation of a protein quaternary structure. Unprimed splenocytes and a B cell lymphoma were capable of presenting only the free (hCG‐α) but not the combined (hCG) α subunit to hCG‐α T cell hybridomas, while hCG‐α‐primed lymph node cells (LNC) responded to both hCG‐α and hCG. As antigen (Ag)‐specific antigen‐presenting cells (APC) present in the hCG‐α‐primed LNC population may be potentially effective for presenting hCG, we investigated the role of specific Ag capture, through mIg and FcγR, in the processing and presentation of hCG and hCG‐a to HAG 5, a T cell hybridoma directed against the immunodominant region (amino acids 61‐81) of hCG‐α. Results showed that only B cells bearing membrane immunoglobulin capable of recognizing hCG‐α and hCG, and present in hCG‐α‐primed mice, were extremely effective in presenting the free as well as the combined a subunit. The effect of FcR‐mediated uptake was analyzed using a B cell line transfected with the FcγRII‐B2 gene to present immune complexes of either hCG‐α or hCG. We found that hCG‐α and hCG were presented equally well, whatever the Ag‐binding site of each antibody to hCG or its a subunit. Using HBG 6, an hCG‐β Tcell hybridoma, we performed similar experiments with the FcγRII‐B2 cell line and determined that the potentiation of hCG presentation to HBG 6 was similar to that observed with HAG 5. Then kinetic experiments were performed to examine the effect of Ag uptake through FcR on processing. Results demonstrated that the uptake pathway drastically influenced the expression of α T cell determinants in the αβ dimer. In addition, treatment with cycloheximide, a protein synthesis inhibitor, only impaired the ability of APC to present specifically captured Ag. Thus, the processing pathway for specifically captured Ag might be different from the pathway used to process nonspecifically captured Ag. This observation might explain why receptor‐enhanced uptake bypasses the inefficient processing of the hCG quaternary structure and enables similar efficiency in the presentation of α and β T cell specificities. These findings provide new insight into the antigenicity of oligomeric molecules, which is modified whether antigen capture is specific or not.
Extensive N nucleotide addition in junctional region of T cell receptor Vγ5 genes rearranged in fetal liver‐derived thymocytes in radiation chimera mice
Authors: Matsuzaki Goro , Ogimoto Masaro , Yoshikai Yasunobu , Seki Ritsuko , Nomoto Kikuo
Published: March 2007

The Vγ5 chain of Tcell receptor γδ is preferentially expressed by murine fetal thymocytes. The fetal Vγ5 gene is known to be homogeneous and lacks N nucleotide addition.We previously reported that Vγ5+ cells were detected among donor‐derived thymocytes from irradiated C3H/He mice soon after reconstitution with AKR/J fetal liver (FL) cells, but that only a few Vγ5+ were detected among donor‐derived thymocytes from irradiated C3H/He mice reconstituted with adult bone marrow (ABM) cells. The results suggest that preferential use of Vγ5 is determined at the level of Tcell precursor generation. In the present report, we analyzed whether the junctional region of FL‐derived Vγ5 genes in the FL chimeras is of fetal type. Sequencing analysis showed that Vγ5 genes from FL chimeras contained extensive N nucleotide addition and were diverse both in nucleotide sequences and deduced amino acid sequences. Vγ5 genes from donor‐derived thymocytes of ABM chimeras, which by polymerase chain reaction were revealed to be less frequent than those of FL chimeras, also showed extensive N addition. Furthermore, the terminal deoxynucleotidyl transferase (TdT) gene, which encodes an enzyme that adds N nucleotides into the junctional region, was expressed at high level in the donor‐derived thymocytes of FL chimeras and normal 8‐week‐old AKR/J thymocytes but at low level in day 17 fetal AKR/J thymocytes. Our results suggest that the preferential rearrangement of the Vγ5 gene is determined at the level of Tcell precursor generation but the homogeneous Vγ5 sequence of fetal type is generated only in the fetal thymic microenvironment where TdT gene expression is low or absent.

The nomenclature of murine TcR γ chains is according to Reilly et al. (Nature 1986. 321: 878). The relationship between the different nomenclature systems is summarized in Takagaki et al. (J. Immunol. 1989. 141: 2112).
Major histocompatibility complex class I molecules are required for the development of insulitis in non‐obese diabetic mice
Authors: Katz Jonathan , Benoist Christophe , Mathis Diane
Published: March 2007

An early step in the development of autoimmune diabetes is lymphocyte infiltration into the islets of Langerhans of the pancreas, or insulitis. The infiltrate contains both CD4+ and CD8+ T cells and both are required for progression to diabetes in non‐obese diabetic (NOD) mice. It has been thought that the CD4+ lymphocytes are the initiators of the disease, the islet invaders, while CD8+ cells are the effectors, the islet destroyers. We question this interpretation because NOD mice lacking MHC class I molecules, hence CD8+ T cells, do not display even insulitis when expected.
Linomide inhibits programmed cell death of peripheral T cells in vivo
Authors: Gonzal José Angel , Gonźlez‐Garciá Ana , Kalland Terje , Hedlund Gunnar , Martínez‐A Carlos , Kroemer Guido
Published: March 2007

Programmed cell death (PCD) is involved in the physiological regulation of lymphocyte turnover, as well in the antigen‐driven selection of T and B cells. Here it is shown that the immunomodulator linomide (quinoline‐3‐carboxamide) inhibits the apoptotic decay of peripheral T lymphocytes in response to three different stimuli. First, linomide reduces the superantigen‐mediated apoptosis and deletion of specific T lymphocytes of both the CD4+ and the CD8+ subsets without affecting other superantigen‐triggered phenomena such as T cell expansion and anergy. Second, linomide abolishes the T lymphopenia and inhibits PCD of splenic CD4+ and CD8+ T cells induced by exogenous glucocorticoids. This effect is restricted to peripheral T lymphocytes and does not concern thymocytes. Finally, linomide abolishes the development of lymphopenia that follows infection with vaccinia virus, while reducing PCD of CD4+ and CD8+ peripheral T cells. The anti‐apoptotic effect of linomide could account for its immunostimulatory properties and might be relevant to the treatment of immunodeficiencies associated with an increased apoptotic decay of T lymphocytes.
Humoral immune response to 2,4‐dinitrophenyl ‐‐ keyhole limpet hemocyanin in antigen‐free, germ‐free and conventional BALB/c mice
Authors: Bos Nicolaas A. , Ploplis Victoria A.
Published: March 2007

The B cell immune response to 2,4‐dinitrophenyl (DNP) keyhole limpet hemocyanin was compared in antigen‐free, germ‐free and conventional BALB/c mice. The numbers of total and of DNP‐specific IgM‐, IgG‐ and IgA‐secreting cells in the spleen were determined by enzyme‐linked immunosorbent plaque assays after primary, secondary and hyperimmunization. Three days after primary immunization a peak of DNP‐specific IgM‐secreting cells was seen in conventional mice only. However, this specific response was accompanied by a rise in the total number of IgM‐secreting cells. At day 6 after primary immunization the total number and the frequency of DNP‐specific IgG‐secreting cells were higher in antigen‐free mice, compared to germ‐free and conventional mice. After secondary immunization in conventional mice only, a considerable bystander IgG response was seen together with the DNP‐specific IgG response. At the end of the secondary response 90% of all IgG‐secreting cells were DNP specific in antigen‐free mice, while the corresponding figure in germ‐free and conventional mice was 63% and 14%, respectively. After hyperimmunization, the absolute number of DNP‐specific IgG‐secreting cells in the spleen was 5‐fold and 11‐fold higher in antigen‐free mice then in germ‐free and conventional mice, respectively. Approximately 48% of all IgG‐secreting cells were DNP specific in antigen‐free mice, while the corresponding figure in germ‐free and conventional mice was 17% and 12%, respectively. The results show that the exogenous antigenic load of animals influences the immune response to newly introduced antigens. The higher absolute and relative numbers of antigen‐specific IgG‐secreting cells after hyperimmunization in antigen‐free mice compared to germ‐free and conventional mice may provide a better source for antigen‐specific B cells that eventually can be used for hybridoma production.
On the biological origin of anti‐double‐stranded (ds)DNA antibodies: systemic lupus erythematosus‐related anti‐dsDNA antibodies are induced by polyomavirus BK in lupus‐prone (NZBxNZW) F1 hybrids, but not in normal mice
Authors: Fredriksen Knut , Osei Awuku , Sundsfjord Arnfinn , Traavik Terje , Rekvig Ole Petter
Published: March 2007

We have recently demonstrated that polyomavirus BK and isolated BK double‐stranded (ds)DNA have a strong potential for induction of anti‐dsDNA antibodies. Here, data are presented that demonstrate that normal mice (a term used in this report for mice not predisposed to a lupus‐like syndrome) of four different strains responded to both BK virus and BK dsDNA by producing transient antibodies binding preferentially to the viral dsDNA itself. These antibodies did not bind in the Crithidia luciliae assay, and did not seem to be of pathogenic significance, as neither signs of proteinuria nor immunochemical signs of glomerulonephritis developed in these mice. In contrast, 5‐week‐old (NZBxNZW)F1 mice developed strong and persistent anti‐dsDNA antibodies in response to BK virus and BK dsDNA, with similar features to those of anti‐dsDNA antibodies from individuals with systemic lupus erythematosus: they reacted strongly in the Crithidia luciliae assay and cross‐reacted with viral as well as with mammalian dsDNA. Furthermore, persistent proteinuria and glomerulonephritis, with demonstrable heavy mesangial deposits of immune complexes containing IgG anti‐dsDNA antibodies, developed 2‐‐3 months earlier than in spontaneously autoimmune control mice. The relevance of these observations to a viral origin of anti‐dsDNA antibodies in lupus is discussed.
Tyrosine phosphorylation of α tubulin in human T lymphocytes
Authors: Ley Steven C. , Verbi Winston , Pappin Darryl J. C. , Druker Brian , Davies Adelina A. , Crumpton Michael J.
Published: March 2007

N‐terminal sequencing of the 55‐ and 50‐kDa polypeptides affinity purified on a phosphotyrosine monoclonal antibody column from activated Jurkat T cells identified α and β tubulin. Two‐dimensional gel analysis indicated that α tubulin was directly phosphorylated on tyrosine. β Tubulin was not detectably tyrosine phosphorylated but was precipitated by anti‐phosphotyrosine (PTyr) antibody by virtue of its association with the α subunit as a heterodimer. Phosphotyrosyl α tubulin was not incorporated into intact microtubules and was all in the unpolymerized soluble fraction. These results suggest that tyrosine phosphorylation of α tubulin may inhibit the ability of this subunit to polymerize into microtubules. Stimulation of Jurkat T cells via T cell receptor increased the amount of tubulin precipitated by the anti‐PTyr antibody. These data raise the possibility that the polymerization of tubulin heterodimers may be regulated by phosphorylation on tyrosine during T cell activation.
Th1 and Th2 CD4+ T cell clones specific for Plasmodium chabaudi but not for an unrelated antigen protect against blood stage P. chabaudi infection
Authors: Taylor‐Robinson Andrew W. , Phillips R. Stephen
Published: March 2007

The host protective immune response to blood stage malaria infection was studied using Plasmodium chabaudi chabaudi (P. chabaudi) in NIH mice. It has been shown previously that CD4+ cells are critically required for protection against erythrocytic infection. Mice lacking a functional CD4+ cell compartment suffer unremitting patent primary parasitemias for at least 60 days after infection. Here, we report that the adoptive transfer of eight P. chabaudi‐specific CD4+ T cell clones of either the Th1 or Th2 type to mice rendered CD4‐depleted by adult thymectomy and anti‐CD4 monoclonal antibody therapy fully restored the ability of recipients to control challenge infection. Control Th1 and Th2 clones specific for an unrelated antigen, ovalbumin, were unable to confer a comparable level of protection in CD4‐depleted mice, even though they received regular doses of the antigen. These data demonstrate that protective immunity to asexual P. chabaudi parasites can be mediated through immune CD4+ T cell clones of either the Th1 or the Th2 subset.
Activation of peripheral large granular lymphocytes with the serine/threonine phosphatase inhibitor, okadaic acid
Authors: McVicar Daniel W. , Mason Anna T. , Bere Earl W. , Ortaldo John R.
Published: March 2007

The murine polyether fatty acid, okadaic acid, is a potent inhibitor of serine/threonine phosphatases in eukaryotic cells. This compound inhibits both protein phosphatase 1 (PP1) and phosphatase 2A (PP2A). Here we have examined the potential of okadaic acid as an activator of fresh peripheral CD3‐ large granular lymphocytes (LGL). We demonstrate that overnight exposure of LGL to as little as 1 nM okadaic acid induced an increase in natural killing against the K562 cell line, but does not induce LAK activity. Optimal cytotoxic activation (2‐fold) occurred at 0.01‐‐1.0 nM okadaic acid, with a return to baseline levels at 10‐‐20 nM, and inhibition, likely due to toxicity, at 40 nM. In addition, okadaic acid at doses ⩾20 nM induced LGL but not T cells to produce interferon‐γ. Similar to phorbol esters, overnight incubation with okadaic acid causes a dose‐dependent reduction in expression of the low‐affinity receptor for the Fc portion of IgG (CD16). However, unlike phorbol ester, short‐term (5 min) okadaic acid treatment did not block CD16‐mediated Ca2+ mobilization in LGL. To address the underlying biochemical mechanisms of okadaic acid activities, the levels of several as‐yet‐unidentified serine/threonine kinases were assayed after renaturation. Under these conditions, okadaic acid induced similar increases in kinase levels in both T cells and LGL. Taken together, these data suggest an important role for PP1 and PP2A in LGL physiology, and define okadaic acid as a potentially important biological response modifier for the study of LGL and Tcell biochemistry, signal transduction, and transcriptional regulation.
Generation of chimeric C5a/formyl peptide receptors: towards the identification of the human C5a receptor binding site
Authors: Pease James E. , Burton Dennis R. , Barker Michael D.
Published: March 2007

We employed the polymerase chain reaction to produce a series of chimeric C5a/formyl peptide receptors. Chinese hamster ovary cells transfected with these constructs were tested for their ability to bind C5a. Substitution of three of the extracellular domains of the C5a receptor with the corresponding domains of the formyl peptide receptor abolished C5a binding, whilst replacement of the first extracellular loop of the C5a receptor with that of the formyl peptide receptor had little effect on the affinity of the receptor for C5a. We therefore conclude that this first outer loop of the C5a receptor does not participate in ligand binding, whilst involvement of the other extracellular domains of the receptor cannot be ruled out.
Ultrastructural analysis of thymic nurse cell epithelium
Authors: Penninger Josef , Rieker Theresa , Romani Nikolaus , Klima Joerg , Salvenmoser Willi , Dietrich Hermann , Stössel Hella , Wick Georg
Published: March 2007

Thymic nurse cells (TNC), a paradigmatic cell type of cortical epithelium, are large lymphoid‐epithelial cell complexes of thymocytes enclosed within vacuoles lined by the epithelial cell membrane. TNC express major histocompatibility complex (MHC) class I and class II molecules on their surface and vacuole‐lining membranes at high density and it was suggested that TNC provide an optimal microenvironment for positive selection of T cells. In this report we present electron microscopical data demonstrating that chicken TNC display morphological structures of exocytosis previously shown for hormone‐secreting cells. In TNC, however, exocytosis is restricted to the capillary cleft between the epithelial cell and engulfed thymocytes. Thus, besides physical contact between the epithelial cell and enclosed thymocytes, TNC may additionally influence the development of thymocytes through release of soluble factors in a restricted microenvironment. By employing the 3‐(2,4‐dinitroanilino)‐3‐amino‐N‐methyl‐propylamine technique which at the ultrastructural level detects acidic organelles involved in processing of antigens presented by MHC class II molecules, we also show that TNC contain acidic compartments similar to classical antigen‐presenting cells, i.e. early and late endosomes and lysosomes, albeit in a lower amount than in thymic dendritic cells. This fact provides evidence that TNC not only are capable of antigen presentation but also possess the intracellular machinery for antigen processing.
Involvement of gp130/interleukin‐6 receptor transducing component in interleukin‐11 receptor
Authors: Fourcin Maryvonne , Chevalier Sylvie , Lebrun Jean‐Jacques , Kelly Paul , Pouplard Annick , Wijdenes John , Gascan Hugues
Published: March 2007

The recently cloned interleukin (IL)‐11 displays many biological properties in common with those reported for IL‐6. In order to analyze the nature and the functionality of the IL‐11 receptor we developed a proliferative assay using the human multifactor‐dependent cell line TF1. We showed that a blocking monoclonal antibody GPX7 raised against the gp130/IL‐6 receptor transducing subunit was also able to inhibit the IL‐11‐triggered TF1 line proliferation. In addition, involvement of gp130 in IL‐11 signaling was demonstrated by an induction of the transducing protein phosphorylation in response to IL‐11, as observed for IL‐6. In contrast, the blocking monoclonal antibody B‐R6, which recognized the gp80/IL‐6 binding subunit, failed to interfere with the IL‐11 proliferative signal in the TF1 cell line. Similarly, we did not observe any competition between IL‐6 and IL‐11 for a putative common binding site on the cell surface. These results suggest that the IL‐11 binding component is different from the gp80/IL‐6 receptor. In conclusion, IL‐11, along with IL‐6, leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor, belongs to the same family of cytokines, using gp130 as a transducing protein.
Prenylated proteins and lymphocyte proliferation: Inhibition by d‐limonene and related monoterpenes
Authors: Schulz Stefan , Bühling Frank , Ansorge Siegfried
Published: March 2007

The aim of the present study was to explore the role of post‐translational isoprenoid modification of cellular proteins in the proliferation of human lymphocytes. We here report that treatment of phytohemagglutinin‐stimulated peripheral blood mononuclear cells with monoterpenes including d‐limonene, perillic acid and perillyl alcohol (0.5–5 mM) which selectively inhibit the isoprenylation of 21–26‐kDa proteins resulted in a dose‐dependent inhibition of DNA synthesis. Cell cycle analysis revealed that perillic acid arrested cells in G1 and prevented cells from entering S phase in a manner similar to that induced by the specific 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitor, compactin. However, unlike compactin, the perillic acid‐induced effects on lymphocyte proliferation were not prevented by addition of mevalonate. We also examined the incorporation of [3H]mevalonate into proteins in resting and phytohemagglutinin‐stimulated lymphocytes during the first 30 h of culture. While in unstimulated lymphocytes radioactivity was predominantly incorporated into a cluster of 21–26‐kDa proteins, mitogenic stimulation was associated with a striking increase in [3H]mevalonate incorporation into a protein (≈︁68 kDa) with migration characteristics similar to that of nuclear lamin B. Treatment of phytohemagglutinin‐stimulated lymphocytes with 5 mM d‐limonene, 2.5 mM perillic acid or 1.25 mM perillyl alcohol strongly suppressed [3H]mevalonate‐labeling of proteins to a degree that correlated with the level of DNA synthesis inhibition. These findings suggest that those mevalonate‐derived products required for lymphocyte proliferation may include one or more isoprenylated proteins and that the isoprenylation of these proteins is required for cell cycle progression.
T cell long‐term hyporesponsiveness follows antigen receptor engagement and results from defective signal transduction
Authors: Dubois Patrice M. , Andris Fabienne , Urbain Jacques , Leo Oberdan , Kaufman Marcelle , Shapiro Robert A. , Gilliland Lisa K. , Ledbetter Jeffrey A.
Published: March 2007

T cell receptor (TCR)‐mediated stimulation of T hybridomas leads to cell activation and lymphokine production that is followed by a long‐term hyporesponsiveness. To investigate the biochemical events involved in the induction and maintenance of this antigen receptor hyporesponsiveness or anergy, we have expressed a G protein/PLCβ1‐coupled muscarinic subtype 1 acetylcholine receptor in a murine T cell hybrid. Transfected cells were capable of responding to both muscarinic agonists and TCR ligands by inducing interleukin‐2 secretion that was sensitive to cyclosporin A and dexamethasone. Both receptors induced tyrosine kinase (TK) activity, but muscarinic stimulation did not affect tyrosine phosphorylation of PLCγ1, nor did the TK inhibitor, herbimycin, block muscarinic receptor‐mediated calcium mobilization. These data indicate that in T cells, the muscarinic receptor mediates T cell effector functions by regulating a TK‐independent proximal pathway which later converges with the TCR pathway. Using these cells, we have explored the long‐term consequences of T cell stimulation via antigen or muscarinic receptors. Our results show that hyporesponsiveness specifically follows TCR engagement and appears to result from a defect in the early signal transduction initiated by TCR cross‐linking. A study of TCR‐mediated signaling supports this model by showing that tyrosine phosphorylation and calcium mobilization are deficient in hyporesponsive T cells.
A peptide binding weakly to the major histocompatibility molecule augments T cell responses
Authors: Feng Ming‐Hsien Lin , Lai Ming‐Zong
Published: March 2007

An I‐Ad‐derived peptide PB1 was found to enhance the reactivity of I‐Ad‐restricted T cells. The augmentative effect was not due to the cross‐reactivity of PB1 peptide with antigens. PB1 had no effect on T cells specific for I‐Ab and I‐Ek, nor did PB1 increase the T cell responses to concanavalin A and staphyloccocal enterotoxin B. The strict I‐Ad specificity suggests that PB1 enhances the recognition of antigen‐I‐Ad complex by T cell receptor. PB1 bound to I‐Ad weakly. The augmentative effect could be found on other I‐Ad‐binding peptides in appropriate conditions; however, PB1 was distinct in its prominently augmentative effect on all the I‐Ad‐restricted T cells analyzed. A similar enhancing activity was demonstrated on a synthetic transferrin receptor peptide with minimum affinity for I‐Ad. The unusual enhancing activity of PB1 may thus be attributed to the low I‐Ad binding affinity. It was postulated that the binding of low‐affinity PB1 would not only stabilize I‐Ad structure, but also enhance the binding of other peptides. This was supported by the increased binding of OVA 323‐339 and cI 84–98 to I‐Ad in the presence of PB1. The inclusion of PB1 in the immunization mixture also enhanced T cell responses in vivo, suggesting the possibility of using low‐affinity peptide to promote specific immunity.
Human tonsil, blood and bone marrow in vivo‐induced B cells capable of spontaneous and high‐rate immunoglobulin secretion in vitro: Differences in the requirements for factors and for adherent and bone marrow stromal cells, as well as distinctive adhesion molecule expression
Authors: Brieva José A. , Roldán Ernesto , Rodríguez Carmen , Navas Gloria
Published: March 2007

Human B cells capable of spontaneous IgG secretion are commonly found in circulation and in lymphoid tissues such as tonsil and bone marrow (BM). The present study compares the mechanisms that regulate tonsil, blood and BM B cells capable of spontaneous IgG secretion. The BM cell subset produced IgG during a markedly longer period of time (14 days) than did tonsil and blood cell subsets (2–3 days). Blood and BM, but not tonsil, B cell IgG secretion depended on the presence of adherent cells, as demonstrated by adherent cell depletion and re‐addition experiments. Stromal BM cells supported linear IgG secretion by non‐adherent BM cells for 2 weeks, but were unable to prolong the short‐term IgG secretion by tonsil and blood cells. Different factors induced IgG secretion in each of the three B cell populations as optimal IgG secretion by tonsil, blood or BM cell subsets required either tumor necrosis factor‐α, interleukin‐6 or fibronectin + interleukin‐6, respectively. Finally, these populations also showed differences in the expression of adhesion molecules; the tonsilar cell subset was PNA+/− CD44+ CD49d+ CD49e− Leu‐8+/−, the blood cell subset was PNA− CD44+/− CD49d+ CD49e− Leu‐8+ and the BM cell subset was PNA− CD44+/− CD49d+ CD49e− Leu‐8−. These results suggest that the mechanisms controlling the final differentiation and the expression of adhesion molecules in these B lymphocytes exhibit territorial specificity.
Profound reduction in allergen sensitivity following treatment with a novel allergy vaccine
Authors: Hellman Lars
Published: March 2007

A novel approach is described for the treatment of IgE‐mediated allergic reactions which is based on the induction of a strong anti‐IgE response in the host. Vaccination of ovalbumin‐sensitized rats with constant domains two and three of rat IgE coupled to a heterologous carrier protein resulted in a profound decrease in serum levels of IgE, and later in a nearly complete block of histamine release from mast cells and basophils upon challenge with either a cross‐linking polyclonal anti‐IgE antiserum or a specific allergen.
A novel soluble form of mouse VCAM‐1 is generated from a glycolipid‐anchored splicing variant
Authors: Hahne Michael , Lenter Martin , Jäger Ute , Vestweber Dietmar
Published: March 2007

VCAM‐1 is a cytokine‐induced endothelial adhesion molecule which belongs to the immunoglobulin (Ig) superfamily and mediates the binding of various leukocytes. In addition to the 110‐kDa form of VCAM‐1, we have found four additional glycoproteins on mouse brain‐derived endothelioma cells after stimulation with tumor necrosis factor‐α (TNF‐α), which are recognized by several monoclonal antibodies against VCAM‐1. Biochemical analysis revealed that the two smaller proteins (35 kDa and 37 kDa) are intracellular precursors of the two larger forms (44 kDa and 45 kDa), that the 44 kDa and 45 kDa proteins are glycolipid‐anchored at the cell surface and that they differ in their N‐glycosylation. Most likely they are identical to the recently identified glycolipid‐anchored splice variant of VCAM‐1, since they are recognized by the M3 antiserum which we raised against a peptide from the unique protein domain of this splicing variant. With the help of this antiserum we could show by immunohistology that the corresponding VCAM‐1 protein variant is induced in vivo by lipopolysaccharide (LPS) on endothelium of the mouse. In addition, we found a 42‐kDa soluble form of VCAM‐1 in the serum of LPS‐stimulated mice, which was recognized by the M3 antiserum. This soluble form was undetectable in the serum of unstimulated mice in contrast to the soluble 100‐kDa form of VCAM‐1 which was clearly detected in serum of unstimulated mice and only increased 2–3‐fold upon stimulation with LPS. Thus, only the expression of the 42‐kDa shedded form and not of the 100‐kDa soluble form of VCAM‐1 is strictly dependent on stimulation by LPS.
Differential effects of superantigen‐induced “anergy” on priming and effector stages of a T cell‐dependent antibody response
Authors: Lussow Alexander R. , MacDonald H. Robson
Published: March 2007

The in vitro T cell nonresponsiveness or anergy to restimulation with staphylococcal enterotoxin B (SEB) following the in vivo injection of the superantigen is well characterized. Here we use mice transgenic for a Vβ8.2+ T cell receptor (TcR) (reactive with SEB) to establish a large population of anergic T cells in vivo. As expected, peripheral T cells from the SEB injected transgenic mice failed to proliferate or produce interleukin (IL)‐2 following restimulation with the superantigen in vitro. However, in this system superantigen reactivity could be restored by either addition of exogenous IL‐2, or stimulation with immobilized anti‐TcR antibody. To evaluate the effects of superantigen‐induced anergy in vivo, SEB‐injected or noninjected control transgenic mice were immunized and boosted with the T cell‐dependent antigen tetanus toxin (TT). SEB injection of the Vβ8.2+ transgenic mice 5 days prior to the TT immunization inhibited the anti‐TT antibody response as measured over a 100‐day period, whereas injection of a superantigen which does not interact with the Vβ8.2+ TcR (such as SEA) did not. Furthermore, SEB injection of control nontransgenic mice did not interfere with the induction of a high titer anti‐TT antibody response. In contrast to the inhibition seen when SEB was given prior to TT immunization, injection of transgenics with SEB either after the priming TT immunization or after the recall booster injection did not significantly influence the titers of anti‐TT antibodies produced. These results demonstrate that the establishment of peripheral T cell anergy to superantigens inhibits the specific antigenic priming of helper T cells in vivo, but does not prevent primed T cells from helping B cells to mount an effective antibody response.
B‐T cell interactions modulate inhibitory effects of interleukin‐4 on human B cell proliferation
Authors: Taieb Joelle , Auffredou Marie‐Thérese , Vazquez Aimé
Published: March 2007

In this study, we investigated the role of B‐T cell contacts in interleukin Ia‐mediated inhibition of human B lymphocyte proliferation induced by mitogenic doses of soluble anti‐μ monoclonal antibody (mAb). We show that additional cross‐linking of B cell antigens, using Sepharose beads coated with anti‐μ, anti‐(IL)‐4 mAb (but not soluble mAb) or anti‐CD40 antigen counteracted the inhibitory activity of IL‐4. More importantly, cell contacts between B cells and activated T cells (but not unstimulated T cells) were sufficient to counteract IL‐4‐mediated inhibition of DNA synthesis. In addition, the inhibitory activity of IL‐4 on chronic lymphocytic leukemia B cells stimulated with anti‐μ and IL‐2 was itself reduced by the presence of fixed activated T cells. Our data suggest that a major role for IL‐4 would be to prepare B cells to receive additional mitogenic signals through cell contact interactions with activated T lymphocytes. When such interactions do not occur IL‐4 may block DNA synthesis, preventing uncontrolled B cell proliferation.
CD8+ cytolytic T lymphocytes become infected in vitro in the process of killing HIV‐1‐infected target cells
Authors: De Maria Andrea , Colombini Sandra , Schnittman Steven M. , Moretta Lorenzo
Published: March 2007

In the present study the requirements for in vitro infection of antigen‐specific CD8+ cytotoxic T lymphocytes (CTL) with human immunodeficiency virus –1(HIV‐1) were investigated. CD3+CD8+CD4− HIV‐1 nef‐specific CTL become infected with HIV‐1 after short‐term co‐culture with HLA‐matched HIV‐1‐infected CD20+ B lymphoblastoid cells (B‐LCL) which are specifically killed. Similar results were observed with an allospecific CD8+ CTL population. In addition, co‐culture experiments showed that once infected with HIV‐1, these CD8+ CTL could spread the infection further to uninfected CD4+ lymphocytes. In contrast, CD8+ CTL did not become infected with HIV‐1 when co‐cultured with HLA‐mismatched HIV‐1‐infected B‐LCL which are not killed.

These observations in vitro could have relevance in peripheral lymphoid organs contributing to the progressive decrease of HIV‐specific CD8+ CTL activity that is associated with the progression to AIDS.
MRC OX19 recognizes the rat CD5 surface glycoprotein, but does not provide evidence for a population of CD5bright B cells
Authors: Vermeer Lilian A. , De Boer Nynke K. , Bucci Cecilia , Bos Nico A. , Kroese Frans G. M. , Alberti Saverio
Published: March 2007

To clone the rat CD5 gene we first produced two rat CD5 probes. The probes were obtained by polymerase chain reaction (PCR) on rat genomic DNA using primers designed on conserved regions between mouse and human CD5. The screening of a rat cDNA library at high stringency using these probes resulted in a 1.5‐kb positive clone. The DNA sequence of this clone confirmed its CD5 nature, but the clone appeared to lack part of the 5′ and part of the 3′ end. These missing 5′ and 3′ ends were obtained by PCR on rat thymus RNA. By ligating these PCR products to the original 1.5‐kb CDM8 clone, a full‐length rat CD5 gene was constructed. The full‐length clone showed high identity with mouse and human CD5; however, at the 5′ site of the gene a region of 36 nucleotides is present which is not seen in either mouse or human CD5. We have evidence that this sequence is a normal constituent of the rat CD5 gene: first, it is in frame with the rest of the CD5 coding sequence; second, it does not contain a stop codon; and third, it is also present in the CD5 gene of other rat strains. We transfected the full‐length CD5 construct in COS cells and demonstrated that indeed the CD5 protein is recognized by MRC OX19. Although we showed that CD5 mRNA is present in rat B cells, extensive flow cytometry analysis using MRC OX19 as antibody failed to detect B cells expressing significant levels of CD5 on their cell surface compared to other B cells in any tissue or cell suspension tested from a variety of rat strains. This is in contrast with the mouse where a distinct population of B cells (B‐la cells) can be found expressing more CD5 than the other B cells. Either B‐1 cells are not present in rats or CD5 is not the right phenotypic marker for rat B‐1 cells. It still remains to be investigated whether a population of B cells with functions similar to those of murine B‐1 cells is present in rats.