bluuebunny/crossref_metadata_2025 huggingface dataset:
B‐cell proliferation activity of pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L. and its structural requirement
Authors: Sakurai  , Matsumoto  , Kiyohara  , Yamada
Published: March 2003

Pectic polysaccharide fraction (BR‐2) containing pharmacologically active pectic polysaccharide, bupleuran 2IIc, which was prepared from a medicinal herb, the roots of Bupleurum falcatum L., was administered orally to C3H/HeJ mice for 7 consecutive days. Proliferative responses of spleen cells were enhanced in the presence of the purified pectic polysaccharide, bupleuran 2IIc, but another B‐cell mitogen, lipopolysaccharide (LPS) did not give a similar effect. In vitro studies using spleen cells showed that bupleuran 2IIc also stimulated lymphocytes, depleted of adherent cells or T cells. Bupleuran 2IIc treatment increased subpopulation of CD25+ and surface immunoglobulin M‐positive (sIgM+) lymphocytes. Non‐specific immunoglobulin secretion of spleen cells treated with bupleuran 2IIc was increased according to the culture time, and coexistence of interleukin‐6 (IL‐6) enhanced the secretion more than that of bupleuran 2IIc alone. These results suggest that bupleuran 2IIc proliferates B cells in the absence of macrophages, and the resulting activated B cells are then induced into antibody‐forming cells in the presence of IL‐6. Among the structural region of bupleuran 2IIc, ramified region (PG‐1), which consists of rhamnogalacturonan core rich in neutral sugar chain, showed the potent mitogenic activity suggesting it to be an active site. Mitogenic activity of bupleuran 2IIc was reduced in the presence of antipolysaccharide antibody (antibupleuran 2IIc/PG‐1‐IgG), which recognizes the ramified region of bupleuran 2IIc as the antigenic epitope. Mitogenic activity of bupleuran 2IIc was also reduced by the addition of β‐d‐GlcpA‐(1→6)‐β‐d‐Galp‐(1→6)‐d‐Galp or β‐d‐GlcpA‐(1→6)‐d‐Galp, which are a part of the epitopes of antibupleuran 2IIc/PG‐1‐IgG. These results suggest that the epitopes in bupleuran 2IIc act as active sites of the polysaccharide during mitogenic activity.
FcγRI activation of phospholipase Cγ1 and protein kinase C in dibutyryl cAMP‐differentiated U937 cells is dependent solely on the tyrosine‐kinase activated form of phosphatidylinositol‐3‐kinase
Authors: Melendez  , Harnett  , Allen
Published: March 2003

The human high affinity receptor for immunoglobulin G, FcγRI, in dibutyryl cyclic AMP (dbcAMP)‐differentiated U937 cells, is coupled to the activation of phospholipase C (PLC) and the conventional protein kinase C (PKC) isoforms, α, β, and γ. Here we demonstrate that aggregation of FcγRI activates the tyrosine‐kinase regulated form of phosphatidylinositol‐3‐kinase (PI‐3‐kinase) and that an increase of phosphatidylinositol trisphosphate (PIP3) is essential for the activation and translocation of PLCγ1 in these cells. In addition, activation of the PKC isoforms was ablated by specific inhibitors of PI3‐kinase or by overexpression of a dominant negative p85 subunit of PI3‐kinase. The findings reported here demonstrate that PLCγ1 and PKC activation by FcγRI are downstream of PI3‐kinase, and that in contrast to cytokine primed cells, only the tyrosine‐kinase activated isoform of PI3‐kinase is coupled to FcγRI in dbcAMP‐differentiated cells.
Induction of dendritic cell costimulator molecule expression is suppressed by T cells in the absence of antigen‐specific signalling: role of cluster formation, CD40 and HLA‐class II for dendritic cell activation
Authors: Mclellan  , Heiser  , Hart§
Published: March 2003

Full activation of T lymphocytes by dendritic cells (DC) during antigen presentation is known to require the interaction of several inducible receptor–ligand pairs. We have postulated that the reciprocal activation of DC by T lymphocytes is also important. Potential signalling molecules that might increase the stimulatory capacity of DC during antigen presentation to T lymphocytes were tested using an in vitro model. Fresh human blood DC were cocultured with CD4+ and CD8+ allogeneic or with autologous T lymphocytes plus Staphylococcus superantigen A (SEA). Surprisingly, costimulator expression on DC cocultured with T lymphocytes was reduced in comparison to DC cultured alone. However, the minority (10–30%) of DC clustering with T lymphocytes showed antigen‐specific up‐regulation of the CD40, CD80 and CD86 costimulator molecules, whereas the non‐clustered DC (70–90%) had less up‐regulation than control DC cultured alone and did not respond to antigen‐specific triggering. Monoclonal antibodies (mAb) to CD40 ligand (CD40L) and human leucocyte antigen (HLA)‐DR, but not lymphocyte function‐associated antigen‐1 (LFA‐1), LFA‐3 or HLA‐class I, significantly inhibited the T‐lymphocyte induction of DC costimulator expression. Since HLA‐class II, but not HLA‐class I mAb, inhibited allogeneic T‐lymphocyte‐mediated activation of DC, CD4T lymphocytes appear to be the main subset activating DC in the mixed lymphocyte reaction. Cross‐linking of CD40, but not HLA‐class II, up‐regulated DC or B‐cell costimulator expression. Although direct class II signalling does not appear to play a role in DC activation, antigen‐specific T‐cell recognition contributes via other mechanisms to regulate DC activation.
Cell surface expression and metabolism of major histocompatibility complex class II invariant chain (CD74) by diverse cell lines
Authors: Ong  , Goldenberg  , Hansen  , Mattes
Published: March 2003

We previously described the processing of antibodies to CD74 (the major histocompatibility complex class II‐associated invariant chain, Ii), by B‐cell lymphoma cell lines. These cells expressed relatively low levels of Ii on the surface, but the molecules were rapidly internalized and replaced by new molecules, so that ≈8×106 antibody molecules per cell were taken up per day. We herein report the results of similar studies with other cell types, namely a melanoma, a colon carcinoma, a T‐cell lymphoma and B‐lymphoblastoid cell lines. The melanoma and the carcinoma were treated with interferon‐γ to induce high levels of the antigen. The T‐cell lymphoma, HUT 78, was selected specifically because it was previously reported to lack cell surface Ii, while expressing the molecule intracellularly. However, HUT 78 displayed Ii on the cell surface, as did the other cell lines tested, and catabolism of the antibody was very fast on all of the cell lines. The capacity of four of the cell lines for cumulative antibody uptake was evaluated, using ‘residualizing’ radiolabels, which are trapped within the cell after catabolism of the antibody to which they were conjugated. A high level of uptake was observed in all cases, although there was significant variation between the cell lines. With melanoma SK‐MEL‐37, the total LL1 uptake in 24 hr was nearly 107 molecules per cell and the average turnover time for Ii on the cell surface was 4 min; with carcinoma HT‐29, the total LL1 uptake in 24 hr was ≈106 molecules per cell, and the average turnover time for Ii on the cell surface was 27 min. Based on the cell content of mature class II antigens (αβ), these data suggest that a large fraction, or all, of immature class II molecules (αβIi) reach the cell surface before entering the peptide‐loading compartment, independent of the particular cell type.
Interleukin‐15 strongly inhibits interleukin‐8 and monocyte chemoattractant protein‐1 production in human colonic epithelial cells
Authors: Lügering N. , Kucharzik T. , Maaser C. , Kraft M. , Domschke W.
Published: March 2003

Interleukin‐15 (IL‐15) is a novel cytokine with actions similar to IL‐2 because of common receptor components. Although IL‐15 is expressed in colonic epithelial cells and may regulate epithelial cell function, its effects on these cells are not fully defined. We explored the regulatory effects of IL‐15 on IL‐8 and monocyte‐chemoattractant protein‐1 (MCP‐1) production in the colonic epithelial cell line Caco‐2 as well as in freshly isolated human colonic epithelial cells. IL‐15 was added to intestinal epithelial cells under various culture conditions. Levels of chemokines were determined by enzyme‐linked immunosorbent assay. To determine the elements of the IL‐2/IL‐15R complex involved we used neutralizing antibodies specific for individual receptor chains. IL‐15 down‐regulates IL‐8 and MCP‐1 production in Caco‐2 cells as well as in freshly isolated human colonic epithelial cells in a dose‐dependent manner. Intestinal epithelial cells became more responsive to IL‐15‐induced suppression when activated with greater IL‐1 doses. Strong chemokine suppression was seen when IL‐15 was given prior to, simultaneous with, or after stimulatory agent. Anti‐IL‐2Rγ antibodies efficiently blocked (82% inhibition) the suppression induced by IL‐15, while anti‐IL‐2Rβ antibodies were less effective. The involvement of β‐chain was further suggested by the finding that a mixture of both monoclonal antibodies (mAb) at a suboptimal concentration (1 µg/ml of each mAb) produced a synergistic inhibitory effect on down‐regulation of epithelial chemokine production. These results show that IL‐15 can suppress IL‐8 and MCP‐1 secretion by intestinal epithelial cells. A microenvironment containing high concentrations of IL‐15 may alter the recruitment of neutrophils to enterocytes at least partly by inhibiting IL‐8 and MCP‐1 production.
Suppression of the reactive oxygen intermediates production of human macrophages by colorectal adenocarcinoma cell lines
Authors: Siegert A. , Denkert C. , Leclere A. , Hauptmann S.
Published: March 2003

Although some in vitro studies indicate that macrophages exert cytotoxic responses against tumour cells by production of reactive oxygen intermediates (ROI), no obvious impairment of tumour cell growth is visible in various human malignant tumours, which contain a large number of tumour‐associated macrophages (TAM). We made use of an in vivo‐like co‐culture model of multicellular tumour spheroids of three colon carcinoma cell lines (HRT‐18, HT‐29, CX‐2) and three functionally different phenotypes of human macrophages (27E10, RM3/1, 25F9) to investigate if tumour cells deactivate macrophage cytotoxicity. The production of ROI was measured by a lucigenin‐amplified chemiluminescence assay in a 96‐well‐microplate luminometer. Different capabilities to produce ROI by different macrophage phenotypes were observed. However, independent of the macrophage phenotype and the tumour cell type a significant inhibition of ROI formation was found in co‐cultures after 1 hr, 1 and 2 days. Macrophages were also suppressed by tumour cell supernatants, which contained anti‐inflammatory cytokines transforming growth factor‐β1 (TGF‐β1) and negligible levels of interleukin‐4 (IL‐4) and IL‐10 as shown by enzyme‐linked immunosorbent assay (ELISA). Although recombinant human cytokines TGF‐β1, IL‐10 and IL‐4 inhibited the production of ROI in freshly isolated monocytes, these cytokines had no effect on differentiated macrophage phenotypes, indicating that these cytokines are not involved in mediating tumour‐induced suppression of ROI production by human macrophages.
Expression of cytokine genes in Marek's disease virus‐infected chickens and chicken embryo fibroblast cultures
Authors: Xing Z. , Schat K. A.
Published: March 2003

The role of cytokines in the pathogenesis and immunity of Marek's disease (MD), a herpesvirus‐induced T‐cell lymphoma in chickens, is poorly understood. Two different experiments were used to examine the potential role of particular cytokines in the pathogenesis and immune responses of MD. First, chicken embryo fibroblasts (CEF) were stimulated with lipopolysaccharide (LPS) and/or recombinant chicken interferon‐γ (rChIFN‐γ) and used to develop techniques for examining transcription of IFN‐α, IFN‐γ, inducible nitric oxide synthase (iNOS), interleukin (IL)‐1β, IL‐2, IL‐6 and IL‐8 by reverse transcription–polymerase chain reaction (RT–PCR). Addition of LPS and/or rChIFN‐γ resulted in the up‐regulation of mRNA for iNOS, IL‐1β and IL‐6, while IFN‐γ was up‐regulated by LPS alone. IL‐2 was down‐regulated by the treatments. Second, to determine the effects of Marek's disease herpesvirus (MDV) infection on cytokine transcription in vivo, chickens were infected with MDV at 21 days of age and examined at 7 days post‐infection (p.i.) (exp. 1) or were infected with MDV at 1 day of age and examined from 3 to 15 days p.i. (exp. 2). In MDV‐infected chickens, IFN‐γ transcription was up‐regulated as early as 3 days p.i. until the termination of the experiment at 15 days p.i., while iNOS and IL‐1β were up‐regulated between 6 and 15 days p.i. Infection of 1‐day‐old chicks increased levels of mRNA for IFN‐γ and iNOS between 16‐ and 64‐fold at 9 days p.i. These results suggest that IFN‐γ and iNOS may play an important role in the pathogenesis of MD.
Antibody repertoire development in fetal and newborn piglets, III. Colonization of the gastrointestinal tract selectively diversifies the preimmune repertoire in mucosal lymphoid tissues
Authors: Butler J. E. , Sun J. , Weber P. , Navarro P. , Francis D.
Published: March 2003

Changes in the VH‐region repertoire of isolator piglets reared for 6 weeks under germ‐free (GF) conditions and those colonized (COL) with a defined exclusion flora on the 1st day of life were compared. Although serum immunoglobulin levels were 20–100‐fold higher in COL piglets than GF piglets, an analysis of peripheral blood B cells (PBBs) indicated that: GF and COL piglets used the same four VH genes and two DH segments during the 6‐week period; proportional usage of VH genes and DH segments was the same as in fetal animals; and VH and DH usage did not differ between COL and GF animals. This pattern differed from the PBBs from 6‐week‐old conventional (CONV) piglets. When the sequences of 73 splenic CDR3 segments were analysed, DH usage and mutation frequency were the same in sequences from both 6‐week‐old GF and COL piglets; mutations were infrequent and occurred with the same frequency as in 110‐day fetal spleen. However, the median CDR3 length in COL piglets was shifted upward due to 3′ DH N‐nucleotide additions. Neither COL nor GF animals made specific serum antibodies to phosphoryl choline given parenterally on a T‐cell dependent carrier. In contrast to the near absence of a colonization effect in PBBs and splenic DNA, rearranged variable heavy‐chain gene segments (VDJs) recovered from the DNA of mucosal lymphoid tissues of COL piglets showed pronounced differences from those recovered from GF animals in usage of DHA‐, DHB‐and VHB‐ and in the frequency of point mutation. The mucosal VDJ transcripts and those from the spleen were similarly affected by colonization. This effect on mucosal lymphoid tissue was consistent with the five‐fold selective increase in serum immunoglobulin A (IgA) levels relative to IgM and IgG. Comparison of IgM and IgA transcripts from mucosal tissues suggested that IgA and IgM clones diversify in parallel. Our findings are the first to show that colonization of the gastrointestinal tract of offspring separated from their mothers, differs from ‘conventionalized’ GF animals in that colonization preferentially influences diversification and expansion of the preimmune IgM and IgA repertoire in mucosal lymphoid tissues but not in PBBs and seldom/modestly in VDJs from splenic DNA.
Ageing and the neutrophil: no appetite for killing?
Authors: Butcher S. , Chahel H. , Lord J. M.
Published: March 2003

In the armoury of the immune system developed to combat the various micro‐organisms that could invade the host, the neutrophil forms the first line of defence against rapidly dividing bacteria and fungi. However, as humans age they become more susceptible to infection with these microbes and this has been ascribed to a decline in immune status, termed immune senescence. Here we summarize the literature specifically concerning the attenuation of neutrophil function with age and the possible mechanisms underlying their reduced response to infectious agents.
Increased immunoglobulin A levels in milk by over‐expressing the murine polymeric immunoglobulin receptor gene in the mammary gland epithelial cells of transgenic mice
Authors: De Groot N. , Van Kuik‐Romeijn P. , Lee S. H. , De Boer H. A.
Published: March 2003

The polymeric immunoglobulin receptor (pIgR) transports dimeric immunoglobulin A (dIgA) across the epithelial cell layers into the secretions of various mucosal and glandular surfaces of mammals. At these mucosal sites, such as the gastrointestinal tract, respiratory tract, urogenital tract and the mammary glands, dIgA protects the body against pathogens. The pIgR binds dIgA at the basolateral side and transports it via the complex mechanism of transcytosis to the apical side of the epithelial cells lining the mucosa. Here, the extracellular part of the receptor is cleaved to form the secretory component (SC), which remains associated to dIgA, thereby protecting it from degradation in the secretions. One pIgR molecule transports only one dIgA molecule (1 : 1 ratio) and the pIgR is not recycled after each round of transport. This implies that the amount of available receptor could be a rate‐limiting factor determining both the rate and amount of IgA transported per cell and therefore determining the total IgA output into the lumen or, in case of the mammary gland, into the milk. In order to test this hypothesis, we set up an in vivo model system. We generated transgenic mice over‐expressing the murine pIgR gene under lactogenic control, by using a milk gene promoter, rather than under immunological control. Mice over‐expressing the pIgR protein, in mammary gland epithelial cells, from 60‐ up to 270‐fold above normal pIgR protein levels showed total IgA levels in the milk to be 1·5–2‐fold higher, respectively, compared with the IgA levels in the milk of non‐transgenic mice. This indicates that the amount of pIgR produced is indeed a limiting factor in the transport of dIgA into the milk under normal non‐inflammatory circumstances.
Polymorphonuclear neutrophils as accessory cells for T‐cell activation: major histocompatibility complex class II restricted antigen‐dependent induction of T‐cell proliferation
Authors: Radsak M. , Iking‐Konert C. , Stegmaier S. , Andrassy K. , Hänsch G. M.
Published: March 2003

Polymophonuclear cells (PMN) of healthy donors do not express major histocompatibility complex (MHC) class II antigens or the T‐cell costimulatory molecules CD80 or CD86. Expression of these receptors, however, is seen in patients with chronic inflammatory diseases. We now report that, by culturing PMN of healthy donors with autologous serum, interferon‐γ (IFN‐γ) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF), de novo synthesis of MHC class II, CD80 and CD86 could be induced. MHC class II‐positive PMN acquired the capacity to present staphylococcus enterotoxin to peripheral T cells, apparent as induction of interleukin‐2 (IL‐2) synthesis and proliferation of the T cells. Moreover, the PMN also processed tetanus toxoid (TT) and induced proliferation of TT‐specific T cells in a MHC class II‐restricted manner. Taken together, these data indicate that PMN can be activated to function as accessory cells for T‐cell activation.
A critical role for alveolar macrophages in elicitation of pulmonary immune fibrosis
Authors: Zhang‐Hoover J. , Sutton A. , Van Rooijen N. , Stein‐Streilein J.
Published: March 2003

Hapten immune pulmonary interstitial fibrosis (HIPIF) is induced by a recall cell‐mediated immune response against the hapten 2,4,6‐trinitrobenzene sulphonic acid (TNBS) in the lung. Studies here dissect the role of the cellular components of the bronchoalveolar lavage (BAL) cells (alveolar macrophages [AMs] versus monocytes and immature dendritic cells) in the fibrogenic inflammatory response. BAL cells from HIPIF mice were generally more activated and produced a greater amount of tumour necrosis factor‐α (TNF‐α) than controls. Liposome‐encapsulated dichloromethylene diphosphonate (Cl2MDP) that was inoculated intranasally (i.n.) into mice selectively depleted AMs. Following AM depletion, the number of TNF‐α‐containing cells was reduced, and both the number of immune inflammatory cells recruited into the alveolar space and the subsequent collagen deposition (hydroxyproline) were decreased in the sensitized and intratracheally (i.t.) challenged mice. In conclusion, AMs are required, in part, for the development of pulmonary fibrosis in HIPIF because AM‐derived factors such as TNF‐α are needed for initiation of chemokine and cytokine pathways and accumulation of immune inflammatory cells.
Molecular cloning, genomic organization and cell‐binding characteristicsof mouse Spα
Authors: Gebe J. A. , Llewellyn M.‐B. C. , Hoggatt H. , Aruffo A.
Published: March 2003

Several group B scavenger receptor cysteine‐rich (SRCR) proteins have been shown to function as modulators in the immune response. Recently, we reported the cloning of a new member of this family, human Spα (hSpα). Herein we report the cloning and characterization of the mouse homologue of hSpα. Like its human counterpart, mouse Spα (mSpα), is a secreted protein containing three SRCR domains. Most lymphoid tissues express RNA transcripts encoding mSpα. Characterization of a genomic clone encoding the mature mSpα protein showed that each of the SRCR domains of mSpα is encoded by a single exon. Comparison of the sequence of mSPα with those of other published proteins indicates that it is the same as the recently reported protein named AIM (apoptosis inhibitor expressed by macrophages). Cell‐binding studies with a mSpα immunoglobulin (mSpα‐Rγ) fusion protein indicated that mSpα is capable of binding to spleen‐derived CD19+ B cells and minimally to peritoneal cavity‐derived CD19+ B cells but not to peripheral blood‐derived B cells. Spleen‐derived CD3+ T cells also bound mSpα‐Rγ; however, no binding was observed to either peripheral blood mononuclear cells or peritoneal cavity‐derived CD3+ T cells. The mSpα‐Rγ fusion protein was also shown to bind to the mouse cell lines WEHI3 (monocytic) and EL‐4 (thymoma, T cell). The cloning of cDNA and genomic clones encoding mSpα and the identification of cells expressing a putative mSpα receptor(s) should facilitate in vivo studies designed to investigate the function of Spα in the immune compartment.
The regulation of prostaglandin output from term intact fetal membranes byanti‐inflammatory cytokines
Authors: Brown N. L. , Alvi S. A. , Elder M. G. , Bennett P. R. , Sullivan M. H. F.
Published: March 2003

Prostaglandins are some of the main mediators which control parturition, and their production by intrauterine tissues can be up‐regulated by pro‐inflammatory cytokines. Anti‐inflammatory cytokines may oppose these effects, and in this study we have investigated how two such cytokines affected fetal membrane function. Interleukin‐10 (IL‐10) inhibited the output of prostaglandin E2 (PGE2) from intact fetal membranes under basal and lipopolysaccharide (LPS)‐stimulated conditions, and there was a parallel decrease in the expression of mRNA for COX‐2. IL‐10 also inhibited the production of interleukin‐1β (IL‐1β) and the expression of mRNA for IL‐1β, indicating that this cytokine has a broad anti‐inflammatory effect. Transforming growth factor‐β1 (TGF‐β1), which is generally considered to be anti‐inflammatory had opposite effects on PGE2 production, in that it increased the output of PGE2 for up to 8 hr. TGF‐β1 increased levels of type‐2 cyclo‐oxygenase (COX‐2) and cytosolic phospholipase A2 (cPLA2) protein, and also activated the cPLA2 enzyme present; the profile of effects is similar to that of the pro‐inflammatory cytokine IL‐1β, and was not expected. Combinations of TGF‐β1 with IL‐1β also increased PGE2 output and caused appropriate changes in prostaglandin pathway enzymes, whereas TGF‐β1 and IL‐1α had more limited effects. Further studies are needed to establish the physiological significance of these findings, but TGF‐β1 does not seem to act as an inhibitory cytokine in intact fetal membranes at term.
Human invariant Vα24+ natural killer T cells activated by α‐galactosylceramide (KRN7000) have cytotoxic anti‐tumour activity through mechanisms distinct from T cells and natural killer cells
Authors: Nicol A. , Nieda M. , Koezuka Y. , Porcelli S. , Suzuki K. , Tadokoro K. , Durrant S. , Juji T.
Published: March 2003

Human Vα24 + NKT cells, a subpopulation of natural killer cell receptor (NKR‐P1A) expressing T cells with an invariant T‐cell receptor (TCR; Vα24JαQ) are stimulated by the glycolipid, α‐galactosylceramide (KRN7000), in a CD1d‐dependent, TCR‐mediated fashion. Little is known about Vα24 + NKT‐cell function. The murine counterpart, Vα14 + NKT cells, appear to have an important role in controlling malignancy. There are no human data examining the role of Vα24 + NKT cells in controlling human malignancy. We report that Vα24 + NKT cells have perforin‐mediated cytotoxicity against haemopoietic malignancies. Vα24 TCR, CD1d and α‐galactosylceramide may all play a role in cytotoxicity but are not absolute requirements. The greatest cytotoxicity was observed against the U937 tumour cell line (95 ± 5% lysis). THP‐1, Molt4, C1R cells and allogeneic mismatched dendritic cells were also sensitive to Vα24 + NKT cytotoxicity but neither the NK target, K562, nor lymphokine‐activated killer‐sensitive Daudi cells, were sensitive. These results indicate a killing pattern distinct from conventional major histocompatibility complex‐restricted T cells, NK cells and other cytotoxic lymphoid cells previously described. We conclude that human Vα24 + NKT cells have cytotoxic anti‐tumour activity against haemopoietic malignancies through effector mechanisms distinct from conventional T cells and NK cells and that their specific stimulator KRN7000 may have therapeutic potential.
An acidic microenvironment impairs the generation of non‐major histocompatibility complex‐restricted killer cells
Authors: Müller B. , Fischer B. , Kreutz W.
Published: March 2003

The microenvironment within solid tumours has often been shown to exhibit an acidic local pH. In recent studies we could demonstrate that an acidic extracellular pH (pHe) inhibits the non‐major histocompatibility complex (MHC) ‐restricted cytotoxicity of immunocompetent effector cells. However, within tumours the activation of cytotoxic cells may already be impaired by low pHe. Therefore, we investigated the influence of acidic conditions on the generation of active killer cells. The cytotoxic activity of natural killer (NK) as well as lymphokine‐activated killer (LAK) cells against K562, Daudi and Raji cells was analysed after an activation period of 3 days at pHe 7·2–6·5. A minor reduction of pHe from 7·2 to 7·0 during the culture period resulted in a strong inhibition of the natural cytotoxicity of NK cells. Furthermore, acidic pHe below 7·2 prevented the generation of activated LAK cells by interleukin‐2 (IL‐2). The cytotoxic capacity could not be reconstituted if cells cultured at a pHe of 6·5 were returned to physiological pH for another 24 hr. Analysis of the cellular subtypes within the various cultures did not reveal differences regarding the frequencies of NK cells, CD8+ T cells, or CD4+ T cells. However, an acidic pHe clearly inhibited the activation‐induced increase of relevant adhesion molecules. The production of cytokines which are involved in the regulation of the cytotoxic process (tumour necrosis factor‐α, interferon‐γ, IL‐10, IL‐12 and transforming growth factor‐β1) was also affected by pHe, as their release was strongly inhibited at pHe 7·0. Furthermore, we observed a considerable decrease in the metabolic activity of effector cells at acidic pHe. In summary, our findings suggest that an acidic microenvironment impairs the induction of an anti‐tumoral immune response within solid tumours.
Gene gun‐mediated delivery of an interleukin‐12 expression plasmid protects against infections with the intracellular protozoan parasites Leishmania major and Trypanosoma cruzi in mice
Authors: Sakai T. , Hisaeda H. , Nakano Y. , Ishikawa H. , Maekawa Y. , Ishii K. , Nitta Y. , Miyazaki J. , Himeno K.
Published: March 2003

An interleukin‐12 (IL‐12) expression plasmid was transferred, using a gene gun, to mice infected with Leishmania major or Trypanosoma cruzi. Transfer of the IL‐12 gene to susceptible BALB/c mice resulted in regression of lesion size and reduced the number of parasites in draining lymph nodes (LN) at the site of L. major infection. Coincident with these protective effects, the T‐helper type (Th) response shifted towards Th1, as evaluated by cytokine production in vitro and L. major‐specific antibody responses. Protective effects of the IL‐12 gene were also observed in T. cruzi infection. Treatment of BALB/c mice infected with T. cruzi enhanced the production of interferon‐γ (IFN‐γ) by spleen cells, while suppressed production of interleukin‐10 (IL‐10) compared with control mice. Administration of anti‐CD4 or anti‐CD8 monoclonal antibody (mAb) abolished the protective immunity against T. cruzi infection, and treatment with the IL‐12 gene could not restore the resistance in these mice. Mice depleted of natural killer (NK) cells with anti‐asialo GM1 also became susceptible to infection, while the resistance was restored when these mice were treated with the IL‐12 gene. Thus, target cells for the treatment appear to be CD4+ and CD8+ T cells, which are ordinarily activated by NK cells. These results suggest that the transfer of cytokine genes using a gene gun is an effective method for investigating the roles of cytokines and gene therapy in infectious diseases.
Fibronectin synthesis by activated T lymphocytes: up‐regulation of asurface‐associated isoform with signalling function
Authors: Wagner C. , Bürger A. , Radsak M. , Blum S. , Hug F. , Hänsch G. M.
Published: March 2003

Fibronectin (FN) is a major constituent of the extracellular matrix. We now provide evidence for a surface‐associated isoform of FN that is synthesized by T cells upon activation. The T‐cell‐derived FN has an unusual splice pattern: an additional domain, EDB, is produced whereas sequences within another domain, IIICS, are spliced out. CS1, the binding domain for very late antigen‐4 (VLA‐4), however, is still generated. To study the potential function of surface‐associated FN its synthesis was down‐regulated by an antisense oligonucleotide, then proliferation of T cells was induced by cross‐linked anti‐CD3. Proliferation was reduced as was expression of CD25. Moreover, when T cells were cultured in high density, the synthetic peptide QILDVPST, corresponding to CS1, inhibited proliferation, as did antibodies to VLA‐4. We propose that surface‐associated FN is a ligand for VLA‐4, which by binding to VLA‐4 on an adjacent cell, provides a costimulatory signal, thus sustaining T‐cell proliferation.
Thymus‐dependent modulation of Ly49 inhibitory receptor expression on NK1.1+γ/δ T cells
Authors: Hara T. , Nishimura H. , Hasegawa Y. , Yoshikai Y.
Published: March 2003

Major histocompatibility complex (MHC) class I‐specific inhibitory receptors are expressed not only on natural killer (NK) cells but also on some subsets of T cells. We here show Ly49 expression on γ/δ T cells in the thymus and liver of β2‐microglobulin‐deficient (β2m−/−) and C57BL/6 (β2m+/+) mice. Ly49C/I or Ly49A receptor was expressed on NK1.1+γ/δ T cells but not on NK1.1−γ/δ T cells. The numbers of NK1.1+γ/δ T cells were significantly smaller in β2m+/+ mice than in β2m−/− mice with the same H‐2b genetic background. Among NK1.1+γ/δ T cells, the proportions of Ly49C/I+ cells but not of Ly49A+ cells, were decreased in β2m+/+ mice, suggesting that cognate interaction between Ly49C/I and H‐2Kb is involved in the reduction of the number of Ly49C/I+γ/δ T cells in β2m+/+ mice. The frequency of Ly49C/I+ cells in NK1.1+γ/δ T cells was lower in both lethally irradiated β2m+/+ mice transplanted with bone marrow (BM) from β2m−/− mice and lethally irradiated β2m−/− mice transplanted with BM from β2m+/+ mice than those in adult thymectomized BM‐transplanted chimera mice. These results suggest that reduction of Ly49C/I+ NK1.1+γ/δ T cells in β2m+/+ mice is at least partly due to the down‐modulation by MHC class I molecules on BM‐derived haematopoietic cells or radioresistant cells in the thymus.
Protection of turkeys against Chlamydophila psittaci challenge by parenteral and mucosal inoculations and the effect of turkey interferon‐γ on genetic immunization
Authors: Vanrompay Daisy , Cox Eric , Kaiser Pete , Lawson Shelly , Van Loock Marnix , Volckaert Guido , Goddeeris Bruno
Published: March 2003

Plasmid DNA (pcDNA1::MOMP A) expressing the major outer membrane protein of an avian Chlamydophila psittaci serovar A strain was tested for its ability to induce protective immunity against challenge with the same C. psittaci serovar. A combined parenteral (intramuscular injection) and mucosal route (DNA drops administered to the nares) of DNA inoculation was compared to three other, different routes of administration (intramuscular inoculation, DNA drops administered to the nares and aerosol immunization). In addition, the effect of turkey interferon gamma (tIFN‐γ) on intramuscular immunization was evaluated by co‐expressing pCIneo::tIFN‐γ. A significant level of protection was observed in turkeys immunized via the combined parenteral/mucosal route, the intramuscular route or by aerosol. Severe clinical signs and lesions were observed in the non‐vaccinated control groups, in 80% of turkeys inoculated with a mixture of pcDNA1::MOMP A and pCIneo::tIFN‐γ, and in 60% of turkeys vaccinated with DNA drops administered to the nares. The use of MOMP‐based DNA vaccination as a means of preventing severe clinical signs and lesions in a turkey model of C. psittaci infection was demonstrated, as was down‐regulation of the immune response by co‐expression of tIFN‐γ.
Interleukin‐12 primes CD4+ T cells for interferon‐γ production and protective immunity during Mycobacterium avium infection
Authors: Silva Regina A. , Flórido Manuela , Appelberg Rui
Published: March 2003

Interleukin‐12 (IL‐12) is a crucial cytokine for the generation of a protective immune response against Mycobacterium avium infection. In contrast to infected control mice, IL‐12‐deficient mice were unable to control bacterial proliferation and their spleen T cells were almost unresponsive in vitro to specific antigens of M. avium. Susceptibility of mice deficient in IL‐12 was similar to that of interferon‐γ (IFN‐γ)‐deficient mice. These data indicate a crucial role of IL‐12 in the development of a T‐cell population able to produce IFN‐γ and to mediate protection against M. avium infection. Treatment of M. avium‐infected mice with IL‐12 induced CD4+ T cells with enhanced capacity to produce IFN‐γ as well as to confer increased protection against M. avium.
Characterization of equine E‐selectin
Authors: Hedges Jodi F. , Demaula Christopher D. , Moore Brian D. , Mclaughlin Bridget E. , Simon Scott I. , Maclachlan N. James
Published: March 2003

Expression of E‐selectin on activated endothelium is a critical initial step that leads to extravasation of leucocytes during inflammation, yet E‐selectin is largely uncharacterized in several animal species including the horse. We have sequenced and compared E‐selectin genes derived from activated cultures of purified equine (horse), cervid (black‐tailed deer) and ovine (sheep) pulmonary artery endothelial cells (ECs). Phylogenetic and amino acid sequence comparisons indicate that bovine, cervid and ovine E‐selectin are similar, whereas human and equine E‐selectin are more closely related to each other than to the ruminant molecules. Human E‐ and P‐selectin‐specific monoclonal antibodies that also recognize equine E‐selectin were identified and used to characterize its expression. Expression of E‐selectin was more readily induced by lipopolysaccharide treatment in equine ECs than in human ECs and supported adhesion and activation of neutrophils, consistent with the extreme sensitivity of horses to endotoxaemia and septic shock.
Suppressive effect of locally produced interleukin‐10 on respiratory syncytial virus infection
Authors: Ruan Yan , Okamoto Yoshitaka , Matsuzaki Zensei , Endo Shuichiro , Matsuoka Tomokazu , Kohno Tadashi , Chazono Hideaki , Eiko Ito , Tsubota Kazuo , Saito Ichiro
Published: March 2003

Interleukin (IL)‐10 is known to be a multifunctional cytokine. This study was designed to evaluate the role of IL‐10 during respiratory syncytial virus (RSV) infection using a C57BL/6 transgenic (TG) mouse model in which the expression of murine IL‐10 cDNA was regulated by a human salivary amylase promoter (IL‐10 TG mice). These mice expressed a large amount of IL‐10 in the nasal mucosa and in salivary glands. Viral replication in the respiratory tract after intranasal infection with RSV was suppressed significantly in IL‐10 TG mice compared to non‐transgenic controls. This suppression was IL‐10 specific, because it was prevented by treating mice with neutralizing anti‐IL‐10 antibodies. We also found that IL‐10‐stimulated T cells displayed cytotoxic activity against infected murine nasal epithelial cells. Previous data indicated that IL‐10 induces Fas ligand (L) expression on mouse T cells. Taken together, these data suggest that Fas/Fas L mediated cytotoxicity is involved in the suppression of RSV replication observed in IL‐10 TG mice after intranasal infection.
Beneficial effect of co‐polymer 1 on cytokine production by CD4 T cells in multiple sclerosis
Authors: Fellay BenoîT , Chofflon Michel , Juillard Catherine , Paunier Anne‐Marie , Landis Theodor , Roth Serge , Gougeon Marie‐Lise
Published: March 2003

Multiple sclerosis (MS) has been associated with an imbalance in the T helper type 1 (Th1) and Th2 subsets. We investigated, at the single‐cell level, the synthesis of pro‐inflammatory cytokines by CD4 and CD8 T cells from MS patients. We report the relationship between priming of CD4 and CD8 T cells for interleukin‐2 (IL‐2), interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) and disease evolution in MS patients, clinically subdivided into relapsing–remitting MS (RRMS) in remission, RRMS in relapse, or chronic progressive MS (CPMS). Moreover, we report the in vivo influence of co‐polymer 1 (COP) treatment on the pattern of cytokine producers in RRMS patients. We show that the frequency of CD4 T cells primed for TNF‐α synthesis increased in all stages of MS, including RRMS remitting, and was normalized to control values in COP‐treated patients (43·2 ± 11·8% in treated patients versus 47 ± 7·3% in RRMS remitting versus 40·3 ± 8% in controls). In addition, a significant decrease in the frequency of CD4 T cells primed for IL‐2 was found in COP‐treated patients as compared to the other groups of patients, reaching values below that of controls (59·1 ± 9·9% in treated patients versus 70 ± 11·6% in RRMS remitting versus 67·1 ± 7·4% in controls). Unexpectedly, COP‐treated patients also showed a significantly decreased priming for IFN‐γ at the CD4 T‐cell level (9·1 ± 3·4% in treated patients versus 18·8 ± 0·6.4% in RRMS remitting versus 15·4 ± 4·7% in controls), but not at the CD8 T‐cell level. This bystander suppression on the inflammatory cells should be considered in the monitoring of MS patients submitted to COP treatment, in order to evaluate better its clinical efficacy.
Reduced T‐cell receptor CD3ζ‐chain protein and sustained CD3ε expression at the site of mycobacterial infection
Authors: Seitzer Ulrike , Kayser Klaus , Höhn Hanni , Entzian Peter , Wacker Hans‐Heinrich , Ploetz Sabine , Flad Hans‐Dieter , Gerdes Johannes , Maeurer Markus J.
Published: March 2003

Control of mycobacterial infection by the cellular immune system relies both on antigen‐presenting cells and on T lymphocytes. The quality of an effective cellular immune response is dependent on functional signal transduction residing in the cytoplasmic tails of the T‐cell receptor CD3 components. In order to investigate potential effects of mycobacteria on T‐cell receptor signalling, we examined the protein expression of T‐cell signal transduction molecules (CD3ζ, ZAP‐70, p59fyn, p56l ck). In Western blots of peripheral blood mononuclear cells of Mycobacterium tuberculosis infected patients, only the CD3ζ‐chain showed a marked reduction in protein expression. To investigate the situation in situ, immunoenzymatic and immunofluorescence stainings for CD3ε and CD3ζ expression were performed on sections of normal lymphoid tissue, M. leprae infected and sarcoid tissue. CD3ε and CD3ζ expression were similar with respect to intensity, localization and the number of cells stained in normal lymphoid tissue and in sarcoid granulomas. In contrast, the granulomas of M. leprae infected tissues showed a significantly reduced expression of CD3ζ compared to CD3ε. Using double immunofluorescence analysis, virtually no CD3ζ expression could be detected in comparison to the CD3ε expression in the lesions. Apparently, mycobacteria are capable of significantly reducing CD3ζ‐chain expression, which may be restored by cytokines. IL‐2‐enhanced ζ‐chain expression and T‐cell effector functions, defined by interferon‐γ release, in M. tuberculosis‐specific and human leucocyte antigen‐DR restricted CD4+ T cells isolated from granuloma lesions from patients with pulmonary tuberculosis. Because CD3ζ is essential for CD3 signalling and for eliciting T‐cell effector functions, reduced CD3ζ protein expression could result in altered signal transduction and inefficient T‐cell effector functions. Alternatively, reduced CD3ζ‐chain expression may protect T cells from repetitive TCR stimulation associated with anergy or apoptosis.
Synergistic interactions of the two classes of ligand, sialyl‐Lewisa/x fuco‐oligosaccharides and short sulpho‐motifs, with the P‐ and L‐selectins: implications for therapeutic inhibitor designs
Authors: Galustian Christine , Childs Robert A. , Stoll Mark , Ishida Hideharu , Kiso Makoto , Feizi Ten
Published: March 2003

The E‐, L‐ and P‐selectins are carbohydrate‐recognizing cell‐adhesion molecules mediating selective leucocyte recruitment in inflammation. The 3′‐sialyl‐ and 3′‐sulpho‐oligosaccharides of Lewisx (Lex) and Lewisa (Lea) series are bound by them, but for high‐avidity binding of P‐ and L‐selectins to the glycoprotein counter‐receptor known as P‐selectin glycoprotein ligand, PSGL‐1, there is a requirement for sulpho‐tyrosines neighbouring a sialyl‐Lex glycan. The two selectins can also bind 3‐O‐ or 6‐O‐sulphated galacto‐lipids (sulphatides). Here we compare some features of the interactions of P‐ and L‐selectins with a novel lipid‐linked sulpho‐tyrosine probe, and with the sulphatides and neoglycolipids of sialyl‐ and sulpho‐Lex/Lea fuco‐oligosaccharides. The sulpho‐tyrosine probe is bound by both selectins. There are close similarities in the interactions of the two selectins with sulpho‐tyrosine and the sulphatides; the binding is relatively resistant to chelation of calcium ions, in contrast to the absolute requirement of calcium ions with the long fuco‐oligosaccharides, including 6‐sulpho‐sialyl‐Lex. With both selectins, there is striking synergy in binding signals elicited by the two ligand types when presented as equimolar mixtures on a matrix. Thus, there are two operationally distinct binding sites on both L‐ and P‐selectin; and the binding sites for sulphate groups in the two ligand types are probably distinct. When sulpho‐tyrosine and sialyl‐Lex are presented on liposomes, a potent inhibitory activity is generated toward the binding of P‐selectin to HL60 cells, with 50% inhibitory concentration (IC50) values in the nanomolar range. These features of the lipid‐linked ligand analogues, and the simple approach for their display on liposomes, may have applications in designs and screening of selectin inhibitors as anti‐inflammatory compounds.
Endothelial cell and cAMP regulation of T‐cell CD40 ligand: relevance of calcium/calmodulin‐dependent kinase IV signalling
Authors: Nielson Christopher P. , Wingett Denise
Published: March 2003

CD40 ligand (CD40L) expression is now recognized to contribute in the pathogenesis of vascular disease. Because increased CD40L has been associated with myocardial infarction, effects of endothelial cells and cAMP with respect to CD40L regulation may be of clinical relevance. In the present study, endothelial cells are shown to markedly increase CD40L on naïve CD4+ T cells with a more modest effect on memory T cells. Furthermore, the addition of dibutyryl cyclic AMP (dbcAMP) synergistically increased naïve cell CD40L but inhibited memory cell CD40L. Although it has previously been recognized that human endothelial cells can increase T‐cell CD40L, this is the first description of the difference in responses of naïve and memory cells and the first demonstration of synergistic effects of endothelial cells and cAMP on CD40L regulation. Consistent with previous reports that CD40L regulation is distinctive, another marker of early activation (CD69) was not similarly regulated. The mechanisms of CD40L regulation were related to calcineurin and calcium/calmodulin dependent kinase IV (CaMKIV) signalling pathways. Endothelial cell costimulation of CD40L was found to be dependent upon calcineurin activity while cAMP actions to increase CD40L were dependent upon CaMKIV. Expression of a dominant negative CaMKIV construct further indicated an important role for CaMKIV in regulation of CD40L and cAMP responses. These data indicate that endothelial cell costimulation can interact with cAMP through calcium signalling pathways to synergistically enhance CD40L expression. Because increased CD40L is associated with atherosclerotic plaque and instability, results are relevant to the pathogenesis of atherosclerosis and myocardial infarction.
Products from human mast cell line cells enhance the production of interferon‐γ by CD8+ and CD4+ T cells
Authors: De Pater‐Huijsen Francina L. , De Riemer MariËlle J. , Reijneke Richard M. R. , Pompen Marjolein , Lutter René , Jansen Henk M. , Out Theo A.
Published: March 2003

In patients with allergic asthma, T‐cell cytokines are implicated in the regulation of the local inflammation in the airways. The ability of sensitized mast cells to release mediators and cytokines early upon allergen stimulation makes them important candidates for local immunoregulation. We have studied the effects of human mast cells on T cells with the use of the human mast cell line HMC‐1. We showed that activated human mast cells or their soluble products induced and enhanced the interferon‐γ (IFN‐γ) production by T cells up to about 60‐fold. The production of interleukin (IL)‐4 was hardly affected and that of IL‐5 was slightly enhanced. The enhancement of IFN‐γ production was induced both in polyclonal CD4+ and CD8+ T cells and in CD4+ and CD8+ T‐cell clones. Further characterization of the factors involved demonstrated a molecular mass above 30 000. Our results implicate that by this mechanism mast cells may account for a negative feedback system locally down‐regulating allergen‐induced T helper 2 responses via IFN‐γ production by the T cells.
Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8
Authors: Micheletti Fabiola , Monini Paolo , Fortini Cinzia , Rimessi Paola , Bazzaro Martina , Andreoni Massimo , Giuliani Massimo , Traniello Serena , Ensoli Barbara , Gavioli Riccardo
Published: March 2003

The human herpesvirus 8 (HHV‐8) is a human γ2‐herpesvirus that is implicated in the development of Kaposi's sarcoma (KS), primary effusion lymphoma and Castelman's disease. Since the responses of cytotoxic T lymphocytes (CTL) play a key role in the control of herpesvirus infection, it is important to identify and to characterize the CTL target epitopes of HHV‐8 viral antigens. In this study, using peptide‐binding motifs, we selected potential human leucocyte antigen (HLA)‐A2‐binding peptides from kaposin A and glycoprotein H (gH), that are latent and lytic HHV‐8 antigens, respectively. HLA‐A2‐binding peptides were tested for their capacity to induce CTL responses in HHV‐8‐negative healthy donors. By this approach, we found that the majority of individuals responded to two HHV‐8‐derived CTL epitopes, namely, VLLNGWRWRL (amino acids 16–25), which derives from kaposin A, and FLNWQNLLNV (amino acids 59–68), which derives from gH. In addition, memory CTL responses to these epitopes were detected in disease‐free individuals infected by HHV‐8 demonstrating that the two epitopes are relevant targets of CTL‐mediated immunity in vivo. The identified epitopes may be investigated for the development of immunotherapeutic strategies against HHV‐8‐associated malignancies.