bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel structures CTLA‐2α and CTLA‐2β expressed in mouse activated T cells and mast cells and homologous to cysteine proteinase proregions
Authors: Denizot François , Brunet Jean‐François , Roustan Paul , Harper Katherine , Suzan Marie , Luciani Marie‐Françoise , Mattei Marie‐Geneviéve , Golstein Pierre
Published: March 2007

Differential screening of a subtracted cDNA library led to the detection of two distinct but homologous mouse cDNA, called CTLA‐2α and CTLA‐2β. The corresponding transcripts have a tissue distribution restricted to T lymphocytes, where they are inducible upon activation, and to mast cells. The open‐frame regions of both cDNA encode proteins homologous to cysteine proteinase precursors, remarkably, however, only to the proregion of these. The ctla‐2α and ctla‐β genes both map to the C1 band of mouse chromosome 13. Sequence comparisons suggest that the proregion of an ancestor proteinase gene evolved to the ctla‐2 genes by successive duplications, first to autonomy, then to amplification. These results raise the question of the possible role of cysteine proteinase proregions, of cysteine proteinases themselves and of inhibitors thereof in activated T lymphocytes; from a different point of view, they also show that some protease proregions may have evolved as autonomous modules.
Expression and characterization of the interleukin 2 receptor in Theileria parva‐infected bovine lymphocytes
Authors: Coquerelle Therese M. , Eichhorn Margarete , Magnuson Nancy S. , Reeves Raymond , Williams Richard O. , Dobbelaere Dirk A. E.
Published: March 2007

We have previously shown that interleukin 2 receptors (IL 2R) are constitutively expressed on the surface of bovine lymphocytes infected with the parasite Theileria parva (Dobbelaere, D. A. E. et al., Proc. Natl. Acad. Sci. USA 1988. 85: 4730). In the present work we characterized these further and showed that IL 2R (Tac antigen) gene expression depended on the continuous presence of the parasite in the host cell cytoplasm. By Northern blot analysis we showed that elimination of the parasite, using a specific theilericidal drug, led to the arrest of Tac antigen mRNA expression. We also investigated receptor internalization and reappearance after receptor‐mediated endocytosis. Binding of human recombinant interleukin 2 (hrIL 2) to the bovine IL 2R caused rapid internalization of the surface IL 2/IL 2R complex. Approximately 50% of 125I‐labeled hrIL 2 was internalized within 10 min. The reappearance of surface IL 2R after ligand‐mediated endocytosis was also studied. Fifty percent of the maximum level of free IL 2R reappeared within 1–1.5 h, but approximately 12 h were needed to restore normal levels of free IL 2R expression after blocking with excess unlabeled hrIL 2.
T lymphocyte heterogeneity in old and young mice: functional defects in T cells selected for poor calcium signal generation
Authors: Philosophe Ben , Miller Richard A.
Published: March 2007

An increase in cytoplasmic free calcium ion concentration is thought to play a critical role in the entry of resting T lymphocytes into the mitotic cycle. Not all murine T cells, however, generate such a calcium signal when exposed to mitogenic doses of concanavalin A (Con A), and the proportion of nonresponsive cells increases with age in adult mice. Since the frequency of T cells able to generate cytotoxic or lymphokine‐secreting cells in culture also declines in old age, we have speculated that the defect in changes in Ca2+ concentration might underlie this functional deficit. To examine the relationship between functional competence and the calcium signaling pathway, we performed limiting dilution analyses of T cells enriched (by a fluorescence‐based cell‐sorting method) for high or low calcium signal generation. We found that rapid Ca2+ signal generation after Con A exposure did indeed correlate well with functional competence in Con A‐stimulated limiting dilution cultures. Furthermore, selection for Ca2+ signals induced by anti‐CD3 antibody, or even by ionomycin, similarly enriched for the functionally competent set of T cells. The ionomycin result, in particular, suggests that the difference between the reactive and nonreactive sets of T cells is unlikely to result simply from alterations in receptor‐linked signal transduction pathways. We discuss a model in which immune senescence involves an age‐dependent increase in a subset of T cells in which changes in cytoplasmic Ca2+ concentration are relatively difficult to induce by either receptor‐dependent mitogens or ionophores.
Recombinant human interleukin 5 is a selective eosinophil chemoattractant
Authors: Wang Ji Ming , Rambaldi Alessandro , Biondi Andrea , Chen Zhen Guo , Sanderson Colin J. , Mantovani Alberto
Published: March 2007

Human recombinant interleukin (IL) induced migration across polycarbonate filters of human peripheral blood eosinophils. The contribution of chemotaxis vs. chemokinesis was investigated using a checkerboard design with both polycarbonate and nitrocellulose filters. When different cytokine concentrations were seeded above and below the filter, maximal induction of migration required a positive concentration gradient between the lower and upper compartments of the chamber, though some gradient‐independent augmentation of migration occurred. These results indicate that induction of eosinophil migration across filter involves actual chemotaxis. The effect of IL 5 was selective for eosinophils with no effect on neutrophils and monocytes. Conversely, granulocyte‐macrophage colony‐stimulating factor elicited migration of both eosinophils and neutrophils. Thus, human IL 5 is a potent and selective chemoattractant for human eosinophils. Eosinophils are selectively localized in tissues under a variety of physiological and pathological conditions. Locally produced IL 5 may play a role in the selective recruitment of eosinophils from the blood compartment.
T cell responses to fractionated Mycobacterium leprae antigens in leprosy. The lepromatous nonresponder defect can be overcome in vitro by stimulation with fractionated M. leprae components
Authors: Ottenhoff Tom H. M. , Converse Paul J. , Gebre Negussie , Wondimu Assefa , Ehrenberg John P. , Kiessling Rolf
Published: March 2007

Protective immunity against Mycobacterium leprae is dependent on M. leprae‐reactive T lymphocytes. M. leprae‐directed T cell reactivity is high in the localized tuberculoid form of leprosy but specifically absent in the disseminated lepromatous type of the disease.

Two important questions that are relevant for the understanding of the immune response in leprosy as well as for the design of rational immunoprophylaxis and ‐therapy strategies are: (a) what are the antigens that trigger T cell responses in tuberculoid patients and thus protect these individuals from developing lepromatous leprosy and (b) is it possible to restore T cell responsiveness to M. leprae in lepromatous patients by rechallenging the immune system with selected antigens that will trigger help but not suppression? We have addressed these question by directly probing the peripheral T cell repertoire of 10 tuberculoid and 18 lepromatous patients with large numbers of different M. leprae and BCG antigenic components that had been separated on the basis of their relative molecular mass (Mr) by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. This technique allows the identification of T cell‐stimulating antigens independent of the expression of B cell epitopes by these antigens. So far T cell epitopes have only been mapped on M. leprae proteins that had previously been defined by antibodies. Our results show that: (a) tuberculoid patients' T cells responded preferentially to M. leprae and BCG antigens in the lower (i.e. < 70 kDa) Mr range with a peak in the 10–25 kDa range; (b) 6 out of 18 lepromatous patients that did not respond to whole M. leprae responded strongly to isolated M. leprae components; antigens in the lower Mr. range were recognized by five out of these six patients and thus commonly seen by both tuberculoid and lepromatous patients' T cells; however, antigens in the higher Mr range, in particular > 150 kDa, were only recognized by lepromatous patients' T lymphocytes; (c) furthermore, the T and B cell repertoires in leprosy patients are skewed towards different antigenic fractions.
Induction of an activation antigen on human endothelial cells in vitro
Authors: Leeuwenberg Jet F. M. , Jeunhomme Trudi M. A. A. , Buurman Wim A.
Published: March 2007

This study describes the expression characteristics of a cell membrane protein recognized by a monoclonal antibody ENA1, which was obtained by immunizing mice with human umbilical vein endothelial (HUVE) cells cultured with a mixture of interleukin 1 and tumor necrosis factor‐α. The expression of this ENA1 antigen could also be induced by lipopolysaccharide and phorbol esters. Expression was only demonstrated on HUVE cells and human umblical arterial endothelial cells, pretreated with one or with a mixture of these reagents. No expression was detected on human fibroblasts, renal epithelial cells or on mesothelial cells derived from omental tissue, either pretreated or not with the aforementioned inducers of the antigen. Furthermore, no reactivity was observed with either polymorphonuclear cells, peripheral blood lymphocytes or the monocytic cell line U937. Time course experiments revealed that the expression of the ENA1 antigen was time dependent. Maximal expression on HUVE cells was observed after 5 h of incubation with activator, after which a decline in expression occurred.

Induction of expression could be completely blocked by the mRNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide, indicating that de novo synthesis occurs. Other pharmacological reagents tested had no effect on the induction of ENA1 expression. The putative role of the newly described antigen is discussed in relation to the current knowledge of molecules involved in adhesion of immune cells in inflammatory processes.
Antibodies to HLA class I promoter‐binding proteins in sera from patients with systemic lupus erythematosus
Authors: Bouteiller Philippe Le , Auphan Nathalie , Weill Bernard , Lemonnier Franç Ois A.
Published: March 2007

Antibodies directed against human cell nuclear factors (HLA‐F1 and HLA‐F2) that bind to the interferon consensus sequence (ICS) in the promoter region of the HLA‐A2 class I gene can be detected in the serum of some patients with systemic lupus erythematosus (SLE). Such antibodies, in gel‐shift assays, inhibited the formation of the retarded band corresponding to the HLA‐F1/F2‐ICS complex irrespective whether purified factors or crude nuclear extracts were used. This inhibitory effect was due to IgG since the SLE serum activity bound to and could be eluted from a protein A‐Sepharose column. This effect still persisted after absorption of SLE serum on a DNA‐affinity column which removed antibodies directed against protein‐free HLA‐A2‐ICS nucleotide sequence also present in such sera. Western blot analysis further confirmed that SLE serum recognized both HLA‐F1 (65 kDa) and HLA‐F2 (88 kDa) polypeptides, independently of the presence of the HLA‐A2‐ICS nucleotidic sequence, as well as other proteins. These results suggest that human sera from some patients with SLE may be used as a convenient source of antibodies directed against DNA‐binding proteins regulating gene transcription.
Expression and regulation of erythrocyte auto‐antibodies in mice following immunization with rat erythrocytes
Authors: Day Michael J. , Russell Jackie , Kitwood Alison J. , Ponsford Mary , Elson Christopher J.
Published: March 2007

Mice immunized with intact rat red blood cells (RBC) developed serum auto‐antibodies (some of which were mouse specific) to the RBC membrane components spectrin and antigens of 100 and 81 kDa as shown by Western blotting and enzyme‐linked immunosorbent assay as well as RBC surface‐bound autoantibodies detected by the Coombs' test. In order to discover whether these autoantibodies were induced and contolled in similar or different ways, mice were challenged with a variety of rat and mouse RBC preparations. In addition, the ability of recipients given spleen cells from the above donors to generate autoantibody responses to intact rat RBC was measured. It was found that all the autoantibodies were induced in mice challenged with rat RBC ghosts but none following immunization with butanol‐extracted rat RBC ghosts or intact mouse RBC. By contrast, mice injected with mouse RBC ghosts made autoantibodies to spectrin and to the 100‐kDa band. Spleen cells from mice primed with intact rat RBC, rat RBC ghosts or butanol‐extracted rat RBC ghosts curtailed Coombs' autoantibody production of recipient mice challenged with intact rat RBC. Serum from recipients of spleen cells primed with intact rat RBC or the butanol extract generally failed to react with rat or mouse spectrin or with the 81‐kDa band, although antibody was detected to the rat 100‐kDa band. Recipients of rat RBC ghost‐primed spleen cells produced antibody to rat and mouse spectrin and to rat 100‐kDa band but not to mouse 100‐kDa or rat or mouse 81‐kDa bands. Occasionally, suppression of antibody to the rat‐specific 38‐kDa band was observed in recipients of intact rat RBC‐primed spleen cells. It is therefore suggested that regulation of cross‐reactive and mouse‐specific autoantibodies as well as rat‐specific antibodies occurs in an independent, determinant‐specific manner.
One allele [G2m(n)] of the human IgG2 locus is more productive than the other
Authors: Rautonen Nina , Sarvas Heikki , Mäkelä Olli
Published: March 2007

Earlier investigators have shown that a high serum concentration of IgG2 and a high IgG2 antibody response to several polysaccharide antigens is associated with the allotype G2m(n) of the IgG2 subclass. We now show that the total IgG2 concentrations are significantly higher in G2m(n) than in G2m(–n) homozygotes (difference of means was 1.26‐fold, p = 0.002). Furthermore, in G2m(n) heterozygotes, the G2m(n) allele contributed more than the G2m(–n) allele to specific IgG2 antibody responses and to the total IgG2.
T cell activation via the CD2 molecule is associated with protein kinase C translocation from the cytosol to the plasma membrane
Authors: Bagnasco Marcello , Nunes Jacques , Lopez Marc , Cerdan Chantal , Pierres Anne , Mawas Claude , Olive Daniel
Published: March 2007

T cell activation via the CD2 molecule involves phospholipase C and phosphoinositide hydrolysis. Here we demonstrate that the triggering of subclones of the human T leukemia Jurkat cell line by anti‐CD2 as well as anti‐CD3 monoclonal antibodies is able to induce activation (i.e. translocation from cytosol to cell membrane) of protein kinase C (PKC), which is dependent on the formation of 1,2‐diacylglycerol from inositol 4–5‐bisphosphate. The kinetics of PKC translocation parallels the rise in intracellular calcium following both CD2 and CD3 stimulations. These results further demonstrate that CD2 and CD3 activation pathways use similar signal transduction mechanisms.
Expression and regulation of CD5 on in vitro activated human B cells
Authors: Freedman Arnold S. , Freeman Gordon , Whitman James , Segil Jeff , Daley John , Levine Herbert , Nadler Lee M.
Published: March 2007

The T cell‐associated antigen CD5 has been shown to play an important role in the regulation of T cell activation. Monoclonal antibodies directed against CD5 upregulate helper function, and induce interleukin 2 (IL 2) production by mature T cells as well as thymocytes. CD5 is also expressed on subsets of B cells associated with autoantibody production, and CD5+ B cells are present in increased numbers in patients with rheumatoid arthritis and systemic lupus erythematosis. More recently CD5 has been found to be present on human B lymphocytes following in vitro activation with phorbol myristate acetate. To date a similar functional role for CD5 has not to date been demonstrated for B cells. In this study we have shown that structurally similar CD5 molecules are present on activated B cells and T cells. In addition, CD5 on both stimulated B cells and T cells is phosphorylated, which may be important in the function of CD5 following activation. CD5 protein or mRNA was not detected on unstimulated splenic B cells depleted of any CD5+ cells. To investigate the control of CD5 expression, we examined a series of cytokines either alone or in combination for their effect on the induction of CD5. CD5 expression was specifically inhibited by IL 4 but not by the other cytokines tested. This inhibition was very specific as IL 4 did not inhibit the expression of other B cell activation antigens including CD25, B5, T9 and CD23 as well as the pan‐B cell antigen CD20. The addition of other cytokines did not increase or reverse the inhibition of CD5 expression by IL 4. This inhibition was demonstrated by immunofluorescence and flow cytometric analysis. Immunoprecipitation studies of 125I‐labeled activated B cells demonstrated that there was a decrease in cell surface CD5 protein, and not simply inhibition of expression of a particular epitope. Northern blot analysis demonstrated that the expression of CD5 mRNA was markedly inhibited in the presence of IL 4, whereas the induction of the protooncogene c‐myb was unaffected. This suggests that IL 4 inhibits CD5 protein expression on activated B cells by reducing the amount of CD5 mRNA transcription or increasing the degradation of CD5 mRNA. The role of the T cell‐derived lymphokine IL 4 in regulating CD5 expression may be important in the disease states characterized by increased numbers of CD5+ B cells.
Immunity to experimental infection with Leishmania major: generation of protective L3T4+ T cell clones recognizing antigen(s) associated with live parasites
Authors: Müller Ingrid , Louis Jacques A.
Published: March 2007

Exacerbation and resolution of lesions induced by Leishmania major promastigotes are, at least in part, the result of the activity of distinct parasite‐specific L3T4+ T lymphocytes. The present report describes L. major‐specific cloned L3T4+ T lymphocytes capable of transferring substantial protective immunity to normal highly susceptible BALB/c mice. The two protective T cell clones analyzed appear to recognize antigen associated only with live L. major parasites. Therefore, the pattern of antigen reactivity of these protective T cell clones is different from that of the previously described parasite‐specific L3T4+ T cells which contribute to exacerbation of disease. The results presented in this report indicate that the two opposite effects of parasite‐specific L3T4+ T cells on the disease process could be mediated by functionally similar L3T4+ T cells differing in their antigen specificity.
Interleukin 1 and tumor necrosis factor enhance transcription from the SV40 early promoter in a T cell line
Authors: Plaetinck Geert , Combe Marie Claire , Corthésy Patricia , Seckinger Philippe , Nabholz Markus
Published: March 2007

Interleukin 1 (IL 1) and tumor necrosis factor (TNF) increase the expression of a number of T lymphocyte‐specific genes in the T cell hybrid PC60. We show here that human IL 1α and 1β, mouse IL 1α and mouse TNF‐α strongly enhance expression of reporter genes transcribed from the SV40 early promoter in this cell line. We found that IL 1 and TNF each induce up to a 30‐fold increase in the rate of transcription, resulting in a proportional increase of mRNA and protein levels. Induction with IL 1 and TNF is detectable after 1 h and reaches a plateau after about 15 h. Removal of these factors from the culture medium results in complete reversion. Induction with IL 1, but not with TNF, can be inhibited with an inhibitor of IL 1 binding. The effects of both factors are not impaired by the protein synthesis inhibitor cycloheximide, suggesting that they stimulate expression from the SV40 early promoter by activation of preexisting transcription factors.
Membrane‐bound IgM obstructs B cell development in transgenic mice
Authors: Müller Werner , Rüther Ulrich , Vieira Paulo , Hombach Joachim , Reth Michael , Rajewsky Klaus
Published: March 2007

Rearranged immunoglobulin genes encoding either the secreted (m̈s) or membrane (m̈m) form of IgM were introduced into the mouse germ line. We report here the phenotypic analysis of lymphocyte populations in these mice (Tm̈s and Tm̈m). In Tm̈s mice the transgenic m̈ chain is present in serum antibodies. The frequencies of B cells in the various B cell compartments of Tm̈S mice are normal or slightly reduced compared to littermates. In Tm̈m mice, however, B cell (but not T cell) development is severely affected. Spleens of 8‐week‐old Tm̈m mice contain about 3%–8% B cells, increasing to approximately 20% at the age of 8 months. These cells express endogenous IgM. B cells expressing only transgenic m̈m chains are not detectable. In the bone marrow B cells are almost completely depleted. A B cell population characterized by reduced levels of IgD on the cell surface is enriched in the spleen and present at almost normal levels in the peritoneum of Tm̈m mice.
Most anti‐human CD3 monoclonal antibodies are directed to the CD3 ε subunit
Authors: Transy Catherine , Moingeon Philippe E. , Marshall Bruce , Stebbins Christopher , Reinherz Ellis L.
Published: March 2007

The T cell receptor is a molecular complex comprized of a clonally restricted heterodimer (Ti) responsible for specific antigen recognition and a set of invariant CD3 peptides termed γ δ, ε ζ and η. The latter are believed to be involved in transmembrane signaling events given that monoclonal antibodies (mAb) directed to the native CD3 structure can trigger T cell activation. We show here that the vast majority of anti‐human CD3 mAb are directed to an epitope(s) encoded in part or in total by the ε subunit since 15 of 18 independent mAb specifically react with a murine T cell line expressing the human CD3 ε chain at its cell surface. The WT31 mAb is also reactive with this cell line showing that its target epitope, originally assigned to the Ti structure, rather maps to the CD3 ε subunit. These findings suggest that the CD3 ε subunit is the most exposed of the native CD3 structures which are immunogenic and that cross‐linking of the CD3 ε chain by mAb mediates the subsequent T cell activation via the T cell receptor complex.
Emigration of B cells from chicken bursa of Fabricius
Authors: Lassila Olli
Published: March 2007

The extent of emigration of cells from the bursa of Fabricius to the periphery was estimated. Per anum application of fluorescein isothiocyanate to label bursal cells in situ was used. Migrant cells can be visualized on frozen sections or cell suspensions of peripheral organs by their fluorescence. The data show that at 2–3 weeks after hatching about 5% of bursal cells leave the bursa per day. Since the bursal cells divide rapidly, this indicates that the vast majority (95%) of bursal cells die in situ. Cells that leave the bursa are surface IgM positive and go first to peripheral blood and later into B cell areas of spleen, thymus and cecal tonsils. The results are also discussed on the basis of their implication for the generation of antibody diversity in the chicken bursa.
Induction of T cell function via the gp33/27 activation inducer molecule (AIM) requires co‐expression of the CD3/TcR complex
Authors: Rincón Mercedes , Cebrián Marta , Sánchez‐Madrid Francisco , López‐Botet Miguel
Published: March 2007

Monoclonal antibodies specific for a dimeric cell surface activation antigen (gp33/27), preliminary designated as activation inducer molecule (AIM), are capable of triggering interleukin 2 (IL 2) synthesis, IL 2 receptor expression and T cell proliferation when used in conjunction with phorbol esters. We have analyzed the functional relationship between the AIM and the CD3/TcR‐mediated activation pathways. Transient modulation of the CD3/TcR complex in the Jurkat cell line, as well as the stable loss of the CD3/TcR surface expression in variant subclones, determined an inhibition of the IL 2 production triggered by anti‐AIM monoclonal antibody (mAb). In contrast, neither the surface expression of AIM nor the ability to respond to a Ca2+ ionophore were affected. Similar results were observed in peripheral blood T lymphocytes, detecting after CD3 modulation an inhibition of both the IL 2 synthesis and the proliferative response to anti‐AIM mAb. Altogether our data indicate that the activation pathway triggered by anti‐AIM mAb is functionally linked to the expression of the CD3/TcR complex in mature T cells.
CD8+CD11b+ peripheral blood T lymphocytes contain lymphokine‐activated killer cell precursors
Authors: Dianzani Umberto , Zarcone Daniela , Pistoia Vito , Grossi Carlo E. , Pileri Alessandro , Massaia Massimo , Ferrarini Manlio
Published: March 2007

CD3+CD8+ cells, purified from peripheral blood T cells by depletion of CD4+ lymphocytes, were tested for their cytotoxic activity against K‐562 or HL‐60 cells. Freshly prepared cells had no cytotoxic function, but upon exposure to recombinant interleukin 2 (rIL 2) in vitro they acquired a lymphokine‐activated killer (LAK) activity. Fractionation of CD3+CD8+ cells into CD11b+ and CD11b− cells demonstrated that all the cells with the potential of developing LAK cell functions were within the CD8+CD11b+ subset. These cells lacked natural killer cell markers such as CD16 or NKH1, but a proportion of them stained for Leu‐7. Furthermore, they expressed the α/β chains, but not the γ/δ chains, of the T cell receptor, as could be determined by staining with the appropriate monoclonal antibodies. CD8+CD11b+ cells had a large granular lymphocyte morphology, as shown by both cytochemical and electron microscopy analyses. They proliferated in response to IL 2 in vitro and developed cytotoxic functions against a number of natural killer resistant targets. Their response to phytohemagglutinin or pokeweed mitogen was very weak or absent. By contrast, CD8+CD11b− cells failed to generate LAK cells in response to rIL 2, did not show a large granular lymphocyte morphology, but responded vigorously to phytohemagglutinin or pokeweed mitogen.

Purified CD8+CD11b+ cells were cloned by limiting dilution in the presence of rIL 2. The observed cloning efficiency of 19 ± 0.3% indicates that a fraction of the cells only could respond to IL 2. Furthermore, only 50% of the clones obtained had a LAK cell function. These data show that CD8+CD11b+ cells represent a heterogeneous cell population. Nevertheless this cell subset probably represents the major source of LAK cell progenitors within the circulating T cells.
Monoclonal antibody 5.5 reacts with p8,14, a myeloid molecule associated with some vascular endothelium
Authors: Hogg Nancy , Allen Catherine , Edgeworth Jonathan
Published: March 2007

The movement of mononuclear phagocytes and neutrophils from the circulation into tissues is a process which is not completely understood. Monoclonal antibody 5.5 is specific for an 8/14‐kDa molecule known variously as the CF antigen, L1 molecule or MRP8 and 14. We show that this molecule, which will be named p8, 14 in this study, is expressed in all circulating monocytes and neutrophils as an intracellular product (as well as some types of epithelium). Tissue staining patterns suggest that when monocytes and neutrophils adhere to vascular endothelium, they release this molecule onto the associated endothelium. This process occurs with single monocytes and when monocytes form part of an inflammatory infiltrate. Monoclonal antibody 5.5 does not react with cultured endothelial cells even when stimulated with phorbol ester, tumor necrosis factor, interferon‐γ or interleukin 1α providing further evidence that myeloid cells are the source of the p8, 14 in this interactive process. Monocytes which have moved further into such tissues and tissue macrophages in general are monoclonal antibody 5.5 negative, suggesting that the ability to synthesize this molecule may be lost when monocytes leave the circulation and enter tissues. These results indicate that p8,14 plays a role in the interaction between myeloid cells and the vascular endothelium to which they adhere prior to leaving the circulation.
Proliferation of lymphocyte subsets in the adult rat: a comparison of different lymphoid organs
Authors: Westermann JÜRgen , Ronneberg Sabine , Fritz Franz Joachim , Pabst Reinhard
Published: March 2007

Adult, male Lewis rats received a single injection of 5‐bromo‐2′‐deoxyuridine (BrdUrd) i.v. to label proliferating cells in the S phase of the cell cycle. After 1 and 24 h the thymus, bone marrow, blood, spleen, peripheral, cervical and mesenteric lymph nodes as well as Peyer's patches were removed. In cell suspensions surface staining was performed for B, T, T helper (Th) and cytotoxic/suppressor (Tc/s) T lymphocytes by identifying χ light chain, CD5+, CD4+ and CD8+ cells, respectively. On the same slide the DNA label BrdUrd was demonstrated by a monoclonal antibody.

B, T, Th and Tc/s lymphocytes proliferate locally both in central lymphoid organs such as the thymus and the bone marow, and in peripheral lymphoid organs such as the spleen, lymph nodes and Peyer's patches. Within an organ the amount of proliferation among the lymphocyte subsets is similar, differing not more than threefold. Although concerning only a small fraction of cells within the organ, an unexpected finding is the high percentage of BrdUrd‐labeled cells among B lymphocytes in the thymus (3%) and among T lymphocytes in the bone marrow (3%). One day after injection of BrdUrd the thymus contains 25% BrdUrd+ T lymphocytes, while the other organs investigated do not show more than about 2% BrdUrd+ B and T lymphocytes. Many of the newly formed lymphocyte subsets leave their organ of birth within 24 h. Thus the amount of proliferation in the lymphocyte subsets investigated is very similar and the differences between central (thymus and bone marrow) and peripheral lymphoid organs are much smaller than expected.
Characterization of C3a receptor‐proteins on guinea pig platelets and human polymorphonuclear leukocytes
Authors: Gerardy‐Schahn Rita , Ambrosius Dorothee , Saunders Derek , Casaretto Monica , Mittler Christa , Karwarth Gabriele , Görgen Susanne , Bitter‐Suermann Dieter
Published: March 2007

The expression of specific membrane receptors for C3a was determined on guinea pig C3a‐sensitive (gp R+) platelets and human polymorphonuclear leukocytes (hu PMNL). Binding studies with 125I‐labeled C3a from gp or hu sources and Scatchard analysis applied to the binding data revealed the existence of two receptor classes on gp R+ platelets; a high‐affinity class with about 200 binding sites/cell and Kd = 1.7 x 10−9 M, and a relatively low‐affinity class with Kd = 10−8 M and about 500 sites/cell. Hu PMNL express a homogeneous receptor class with Kd = 3 x 10−8 M and 40000 sites/cell. Molecular characterization of the C3a receptor on gp R+ platelets was achieved by (a) cross‐linking photoaffinity‐labeled receptors to bound 125I‐labeled C3a; (b) photoaffinity labeling receptors with a 13‐amino acid residue C3a analogue 125I‐Nap‐Ahx‐13; and (c) use of chemical cross‐linkers like disuccinimidylsuberate to cross‐link receptors with 125I‐C3a. All three techniques gave rise to very similar labeling patterns. With the photoaffinity labeling methods, a diffuse band pattern was observed with an apparent molecular mass of 95–123 kDa with 125I‐C3a as label, and 85–105 kDa with 125I‐Nap‐Ahx‐13 as label. Chemical cross‐linking of 125I‐C3a revealed three distinct bands with molecular masses of approximately 123, 108 and 95 kDa. Subtracting the contribution of the cross‐linked ligands, the C3a receptor on gp R+ platelets appears to be a protein complex, consisting of one to three components with estimated molecular masses between 83–114 kDa.
Immunoglobulin VH gene expression in Ly‐1+ and conventional B lymphocytes
Authors: Andrade Luiz , Freitas António A. , Huetz François , Poncet Pascal , Coutinho António
Published: March 2007

Lymphocyte populations in which Ly‐1 B cells are differentially represented were studied for the expression of ten VH gene families, either by an RNA colony blot assay or by in situ hybridization of single cells, in BALB/c and C57BL/6 mice. The comparisons of cells from lymph nodes, Peyer's patches and adult spleen (poor in Ly‐1 B cells) with cells from peritoneal cavity and neonatal spleen (rich in Ly‐1 B cells) were confirmed by the analysis of adult peritoneal Ly‐1− and Ly‐1+ B cells sorted on the fluorescence‐activated cell sorter. The results indicate that the peritoneal Ly‐1+ B subset uses the whole spectrum of known VH gene families, and shows a preferential utilization of CP12 VH genes, most likely as a result of a selective process during life.
Histone H1° mapping using monoclonal antibodies
Authors: Dousson Sandrine , Gorka Claude , Gilly Claudine , Lawrence Jean‐Jacques
Published: March 2007

Monoclonal antibodies (mAb) to ox liver histone H1° were produced and characterized. Two sets of mice were immunized either with pure H1° or with an H1°‐yeast tRNA complex. Eleven hybridomas of various clonal origin were selected. Typing of the antibodies indicated that all but three IgM belonged to the IgG1 class and contained χ light chains. Immunoblotting experiments using peptides derived from H1° or H5 treated by various proteolytic agents (trypsin, N‐bromosuccinimide, cyanogen bromide, acetic acid), revealed that nine of the mAb reacted with the globular part of H1°. More advanced characterization of the antigenic determinants allowed us to determine distinct regions within this globular part which are involved in the antigenic recognition. The peptopes could be subdivided into two groups. Three mAb bound to residues 24–27 and were specific for H1°. Six mAb bound to residues 27–30 and were specific for H1° except one of them which strongly cross‐reacted with H5 and GH5. Two mAb reacted with the entire histone H1° but failed to react with any of the peptides, suggesting that the corresponding epitope is a conformational antigenic determinant. In order to confirm the localization of the two distinct regions which are involved in the antigenic recognition, a synthetic decapeptide corresponding to the beginning of human H1° globular part (from residue 19 to residue 28) was synthesized. Inhibition experiments of the reaction between H1° and the various IgG1 mAb by increasing amounts of peptide‐bovine serum albumin conjugates were then performed.
Interleukin 2 receptor expression and interleukin 2 production in exponentially growing T cells: major differences between in vivo and in vitro proliferating T lymphocytes
Authors: Rocha Benedita , Lembezat Marie‐Pierre , Freitas Antonio , Bandeira Antonio
Published: March 2007

In the present study we have assessed the growth requirements for in vivo proliferating mature T cells. For that purpose we have selected experimental approaches which allow the study of exponential growth in vivo of a major fraction of T cells, and make it possible to obtain large numbers of T cells in cycle. Two types of growing T cell populations were used: peripheral T lymphocytes, proliferating exponentially after transfer into syngeneic athymic nude mice, and activated T cells in lymph nodes of normal mice draining the site of oxazolone administration. The results obtained show that mature T cell growth in vivo is not accompanied by expression of high‐affinity interleukin 2 (IL2) receptor in the majority of activated cells, is not abrogated by in vivo administration of anti‐IL2 receptor antibodies or enhanced by the in vivo injection of recombinant IL2, and that in vivo growing T cells do not produce detectable amounts of IL2, as evaluated functionally by limiting dilution assays or the presence of IL2 mRNA, detected by Northern blots or in situ hybridization. The presented data thus indicate that the rules known to apply to T cell activation and proliferation in vitro differ from those used by in vivo growing T lymphocytes, at least in the two systems studied.
Human interleukin 6 and tumor necrosis factor α production studied at a single‐cell level
Authors: Andersson Ulf , Matsuda Tadashi
Published: March 2007

Individual peripheral blood mononuclear cells, which produced interleukin 6 (IL 6) or tumor necrosis factor α, (TNF α), were studied by cytokine‐specific polyclonal or monoclonal antibodies (mAb) and immunofluorescence technique with UV microscopy. Lipopolysaccharide (LPS) induced IL 6 as well as TNF α production in the majority of the monocytes, but not at all in lymphocytes. Approximately every second monocyte made TNF α in response to LPS within 0.5 h from start of the cultures, when no IL6 or TNF α production occurred. The maximal number of TNF α‐synthesizing monocytes was observed 1.5 h later and then rapidly declined. LPS stimulation led to optimal IL 6 production 3 h after initiation of the cultures, with 90% of the monocytes expressing intracellular IL 6. LPS‐induced IL 6 synthesis started about 1 h after that of TNF α. Polyclonal T cell activation with staphylococcal enterotoxin A or anti‐CD3 mAb induced a biphasic production pattern of IL 6 as well as TNF α. Early IL 6 synthesis, which peaked 6–8 h from start of the cultures, occurred exclusively in monocytes, while late IL 6 production at 48 h was restricted to a small fraction of lymphoid cells. T cell mitogen induced early TNF α production, which peaked at 6 h, took mainly place in monocytes and to a minor degree in CD4+ as well as CD8+ T cells. The majority of the TNF α‐producing mononuclear cells at 24 h were of the CD4+ T cell lineage in the staphylococcal enterotoxin A‐ or anti‐CD3 mAb‐activated cultures. IL 6 as well as TNF α accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.
Influence of sulfated carbohydrates on the accessibility of CD4 and other CD molecules on the cell surface and implications for human immunodeficiency virus infection
Authors: Thiele Bernhard , Braig Henk Ronald , Ehm Ingo , Kunze Rudolf , Ruf Bernhard
Published: March 2007

The modulatory activity of dextran sulfate with a relative molecular mass of 8 and 500 kDa and pentosan polysulfate with a relative molecular mass of 6 kDa on human T cell surface molecules CD2, CD3, CD4, CD8 and HLA‐DR was investigated by analytical flow cytometry. The 500‐kDa dextran sulfate induces a complete disappearance of the CD4 and a 50% diminuition of CD2 immune reactivity on peripheral blood lymphocytes after a 4‐h incubation while the low molecular mass polyanions do not. This modulation of the CD4 immune reactivity includes all CD4 epitopes investigated. It does not correlate with the antiviral effect of polyanions against human immunodeficiency virus infection. The interaction of polyanions with the CD4 presentation is temperature dependent and differs between fresh lymphocytes and immortal cell lines. From our data it can be concluded that mechanisms other than cell surface effects are responsible for the antiviral potency of these drugs. Implications for the modes of antiviral action of sulfated carbohydrates are discussed.
Characterization of cytoplasmic and nuclear polypeptides induced by interferon‐γ in a murine pre‐B cell leukemia
Authors: Franco Antonella , Fizzotti Marco , Gariglio Marisa , Gribaudo Giorgio , Cavallo Giorgio , Landolfo Santo
Published: March 2007

The effects of interferon‐γ (IFN‐γ) on protein synthesis of a pre‐B cell leukemia, L1210, have been studied by two‐dimensional gel electrophoresis. In total cell extracts, at least ten proteins were induced de novo, or increased in their expression, after an 18‐h IFN‐γ treatment, whereas in the nuclear extracts eight proteins were specifically induced. Of these, increased synthesis of a 40‐kDa/pI 5.9 cytoplasmic protein was the most prominent and reproducible. Most of these proteins appear to be specific for a defined step of differentiation, since they are not found in other B cell leukemias upon IFN‐γ treatment. Others appear to be tissue specific, since they are not induced in fibroblasts nor in T cells. In addition, synthesis of some of the induced proteins appeared to require rapid transcription of new mRNA, because actinomycin D markedly inhibited their formation when added immediately before IFN‐γ. In keeping with this finding, in vitro translation of mRNA from IFN‐γ‐treated L1210 cells into a rabbit reticulocyte lysate system, followed by analysis of the labeled proteins by two‐dimensional gel electrophoresis, revealed the appearance of at least seven proteins.

Taken as a whole, these results demonstrate that in leukemic pre‐B cells IFN‐γ induces the transcriptional activation of genes coding for cytoplasmic and nuclear proteins, some of which could be employed as specific cell activation markers.
Morphologic and functional characterization of human peripheral blood T cells expressing the T cell receptor γ/δ*
Authors: Ferrini Silvano , Zarcone Daniela , Viale Maurizio , Cerruti Giannamaria , Millo Romano , Moretta Alessandro , Grossi Carlo E.
Published: March 2007

The morphologic and functional characteristics of cells freshly isolated from human peripheral blood and bearing a T cell receptor (TcR) γ/δ were analyzed. Cell preparations highly enriched for TcR γ/δ+ cells were obtained by treatment of E rosette‐forming lymphocytes with anti‐CD4 and anti‐CD8 monoclonal antibodies (mAb) and complement. These preparations consisted of 64–82% TcR γ/δ+ lymphocytes, as indicated by the sum of cells reacting with the BB3 and A13 mAb which define two distinct, nonoverlapping, TcR γ/δ+ cell subsets in the peripheral blood. TcR γ/δ cells were able to form conjugates with the natural killer‐sensitive K‐562 and with the natural killer‐resistant HL‐60‐R tumor cell lines. The cytochemical localization of lysosomal acid hydrolases showed that 95%–98% of the cells in the TcR γ/δ+ preparations had the morphologic features of granular lymphocytes. Moreover, electron microscopy analyses showed that TcR γ/δ+ cells had electron‐dense granules dispersed in the cytoplasm and a variety of smooth vesicles, a morphology identical to that of other CD3‐ or CD3+ granular lymphocyte subsets. Freshly isolated TcR γ/δ+ cells were unable to lyse K‐562 and natural killer‐resistant targets, such as HL‐60‐R and P815. However, low levels of target cell lysis were observed upon triggering of the effectors by anti‐CD3 TcR mAb or by lectin. After short‐term culture with inter‐leukin 2, TcR γ/δ+ cells acquired a strong cytolytic activity against K‐562 and HL‐60‐R target cells in the absence of triggering stimuli, and also displayed high levels of cytolytic activity against P815 in the presence of anti‐CD3/TcR mAb.
The chemotactic activity for granulocytes produced by virally infected fibroblasts is identical to monocyte‐derived interleukin 8
Authors: Damme Jo Van , Decock Benny , Conings Renè , Lenaerts Jean‐Pierre , Opdenakker Ghislain , Billiau Alfons
Published: March 2007

So far, the role of fibroblasts in inflammatory processes has been underestimated. We have previously shown that stimulation of fibroblasts with viruses or bacteria results in a simultaneous production of several cytokines, including interferon‐β, interleukin (IL) 6 and colony‐stimulating factors. We here report that virally infected fibroblasts produce also a chemotactic factor for granulocytes. The activity is inducible not only by measles virus but also by IL 1β and the double‐stranded RNA poly(rI) ˙ poly(rC). This factor, when purified to homogeneity, occurs as a 6–7‐kDa protein doublet upon sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The pure protein is serologically related to a fully characterized granulocyte chemotactic peptide (GCP) from monocytes, designated IL8. Furthermore, the chemotactic factor from fibroblasts has an NH2‐terminal sequence identical to that of GCP/IL 8, small differences in NH2‐terminal processing being observed. Finally, in addition to diploid fibroblasts, the osteosarcoma MG‐63 cell line is also a producer of GCP/IL 8. It can thus be concluded that GCP/IL8 can be produced by several cell types in response to infection and that fibroblasts can contribute to chemotaxis in inflammation.
Ontogenic development of autoantibody repertoires in spleen and peritoneal cavity of normal mice: examples of T cell‐dependent and ‐independent reactivities
Authors: Huetz François , Poncet Pascal , Coutinho António , Portnoi Denis
Published: March 2007

The ontogenic development of B cell clonal precursors (BCP) reactive to bromelaintreated, syngeneic erythrocytes (BrMRC) and to single‐stranded DNA has been studied by limiting dilution of both spleen and peritoneal cells. It was found that the frequency of anti‐BrMRC BCP in the spleen is very low up to 4 weeks of age and slowly increases thereafter, to reach adult levels by 6–10 weeks. In the peritoneal cavity, no such BCP can be found before 2 weeks, but they occur at a very high frequency already by 3 weeks of age. Injection of adult, normal syngeneic T cells at birth has no apparent effect on the representation of anti‐BrMRC BCP in the peritoneal cavity, but brings these to adult levels or even higher in the spleen already at 3 weeks of age. Accordingly, adult athymic (nude) mice contain normal frequencies of BrMRC‐specific BCP in the peritoneal cavity but are devoid of such clones in the spleen. In contrast, the frequency of anti‐DNA BCP is very high throughout postnatal development in both spleen and peritoneal cavity, of normal and athymic mice, in both resting and naturally activated splenic B cell compartments, and it is independent of T cell transfers into nude animals. These results indicate the role of T cells in the establishment of some clonal specificities in the adult, splenic autoreactive B cell repertoire.
A novel CD3‐ J11d+ subset of CD4+CD8‐ cells repopulating thymus in radiation bone marrow chimeras
Authors: Matsumoto Koji , Yoshikai Yasunobu , Matsuzaki Goro , Asano Taiji , Nomoto Kikuo
Published: March 2007

Sequential appearance of T cell subpopulations occurs in the thymus of irradiated AKR (H‐2k, Thy‐1.1) mice at an early stage after transplantation with bone marrow cells of C3H/HeN (H‐2k, Thy‐1.2) mice. The donor‐derived thymocytes were first detected on day 8 after bone marrow reconstitution. Although most of the thymocytes were CD4‐CD8‐ cells, an appreciable level of CD4+CD8+ cells was detected in the thymus at this stage. The early appearing CD4+CD8‐ cells were a novel subset of thymocytes that were J11d+CD3‐. From day 10 to day 21 the proportion of CD4+CD8‐CD3‐J11d+ cells decreased while the proportion of CD4+CD8‐ cells and CD4+CD8‐CD3+J11d‐ cells increased. The CD4+CD8‐CD3‐ cells seem to diversify to form CD4+CD8+ thymocytes after short‐term culture in vitro. These results suggested the existence of a differential pathway from CD4‐CD8‐ cells to CD4+CD8+ cells via CD4+CD8‐ cells in the thymus.
Cell surface antigen CD5 is a marker for activated human B cells
Authors: Werner‐Favre Christiane , Vischer Thomas L. , Wohlwend Dominique , Zubler Rudolf H.
Published: March 2007

A minor subset of B cells which in vivo express the surface antigen CD5, has attracted much attention because of its involvement in autoimmune responses. On the basis of observations showing self‐renewal capacity of such cells in mice and also the absence of a substantial change of CD5 phenotype during B cell activation in vitro, the CD5+ B cells are now generally considered to represent a separate cell lineage. In the present study, CD5‐ B cells were isolated by cell sorter and then stimulated in vitro with mutagenized EL4 thymoma cells in the presence of T cell supernatant. About 70% of the B cells were CD5+ after 3 days. Thus, the CD5 antigen behaves as a B cell activation marker. In our system we found that the frequency of rheumatoid factor‐producing B cells was on average three times higher in CD5+ than in CD5‐ B cells isolated ex vivo from human peripheral blood. Most likely this reflects frequent activation of such autoreactive B cells in vivo.