bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential cytokine regulation of natural killer cell‐mediated necrotic and apoptotic cytotoxicity
Authors: GARDINER  , REEN
Published: March 2003

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr‐release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine‐activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I‐deoxyuridine (125I‐UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC‐1 target cells. Interleukin‐4 (IL‐4) inhibited interleukin‐2 (IL‐2)‐induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL‐4 inhibited both adult and cord LAK cytotoxicity of YAC‐1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL‐2 induced higher, and IL‐4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co‐culture of either adult or cord LAK cells with IL‐4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL‐4 did not inhibit perforin expression, thereby defining IL‐4 as a cytokine that can differentially regulate the NK cell‐mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft‐versus‐host disease and in the graft‐versus‐leukaemia phenomena.
Dietary fish oil diminishes lymphocyte adhesion to macrophage and endothelial cell monolayers
Authors: SANDERSON  , CALDER
Published: March 2003

To further investigate the imm_unomodulatory effects of dietary lipids, rats were fed on a low‐fat diet or on high‐fat diets that contained hydrogenated coconut, olive, safflower, evening primrose or fish oil as the principal fat source. The fish oil diet decreased the level of expression of CD2, CD11a, CD18 and CD44 on the surface of freshly prepared lymphocytes and of CD2, CD11a, CD18, CD54 (intercellular adhesion molecule‐1; ICAM‐1) and CD62L (L‐selectin) on the surface of concanavalin A (Con A)‐stimulated lymphocytes. The olive oil diet also resulted in decreased expression of some adhesion molecules. The fish or olive oil diets, and to a lesser extent the safflower or evening primrose oil diets, decreased the adhesion of both freshly prepared and Con A‐stimulated lymphocytes to macrophage monolayers. The fish oil diet, and to a lesser extent the olive or evening primrose oil diets, reduced the ability of Con A‐stimulated lymphocytes to adhere to untreated endothelial cells. Furthermore, the fish oil diet resulted in a 50% reduction in Con A‐stimulated lymphocyte adhesion to tumour necrosis factor‐α (TNF‐α)‐stimulated endothelial cells. This study demonstrates that dietary lipids affect the expression of functionally important adhesion molecules on the surface of lymphocytes. Furthermore, this study suggests that such diet‐induced effects on adhesion molecule expression might alter the ability of lymphocytes to bind to macrophages and to endothelial cells. Of the diets studied fish oil causes the most significant effects. The results of this study suggest that a reduction in cellular infiltration may partly explain the protective effect of a fish‐oil‐rich diet against the development of inflammatory and cardiovascular diseases.
Soluble Fas mRNA is dominantly expressed in cases with silicosis
Authors: Otsuki T. , Sakaguchi H. , Tomokuni A. , Aikoh T. , Matsuki T. , Kawakami Y. , Kusaka M. , Ueki H. , Kita S. , Ueki A.
Published: March 2003

Although it is well known that cases with silicosis exhibit various immunological abnormalities, the mechanisms involved in the occurrence of immuno‐dysfunction or dysregulation induced by silica compounds have not yet been determined. Fas is a well‐known cell surface molecule that is involved in the apoptosis pathway that belongs to the tumour necrosis factor‐receptor family. Soluble Fas (sFas) is produced as an alternatively spliced product of the Fas gene and protects cells from apoptosis due to antagonization of the binding between membrane form of the Fas gene (mFas) and the Fas ligand. To determine the role of the Fas/Fas ligand system in silica‐induced immunological abnormalities, we investigated Fas and Fas‐ligand message expression levels using the multiplex reverse transcription–polymerase chain reaction (RT–PCR) method with peripheral blood mononuclear cells from silicosis cases with no clinical symptoms of autoimmune diseases. Although the relative expression levels of the Fas or Fas‐ligand genes were not remarkably altered in these cases, we observed the sFas message was dominantly expressed compared with mFas expression. These results suggest that self‐recognizing clones in cases with silicosis survive for decades, escaping the exclusion mechanisms induced by apoptosis. Then they cause the appearance of autoantibodies and the acquisition of autoimmune diseases sequentially.

sFas, soluble Fas PSS, progressive systemic sclerosis RT–PCR, reverse transcriptase–polymerase chain reaction mFas, membrane Fas SLE, systemic lupus erythematosus HV, healthy volunteers SQ‐MPL‐PCR, semi‐quantitative multiplex RT–PCR PR, radiological profusion grades.
Potentiation of antigen‐specific, Th1 immune responses by multiple DNA vaccination with an ovalbumin/interferon‐γ hybrid construct
Authors: Lim Y. S. , Kang B. Y. , Kim E. J. , Kim S. H. , Hwang S. Y. , Kim T. S.
Published: March 2003

The preferential differentiation of T helper (Th) cells to Th1 or Th2 subsets is important with respect to susceptibility or resistance to particular infections, or to autoimmune diseases and allergic diseases. To more effectively drive immune responses toward antigen‐specific Th1 responses, we constructed a mammalian expression vector (pOVA/IFN‐γ) carrying a hybrid gene in which the ovalbumin (OVA) (a model antigen) cDNA was covalently linked to murine interferon‐γ (IFN‐γ) cDNA. Intramuscular injection of BALB/c mice with the pOVA/IFN‐γ DNA increased both the production of OVA‐specific IFN‐γ by CD4+ T cells and the ratio of anti‐OVA immunoglobulin G (IgG)2a to IgG1 isotypes, while the injection with the pOVA alone, or with the mixture of the pOVA and pIFN‐γ, caused no or little increase. Furthermore, the OVA‐specific, Th1 immune responses were dramatically augmented by multiple injections with the pOVA/IFN‐γ DNA. These studies indicate that the direct linkage of an OVA gene to an IFN‐γ gene in the expression plasmid is required for efficiently confining the Th1 effects of IFN‐γ to the OVA‐specific cells, and the linkage effect of the OVA/IFN‐γ DNA can be potentiated by multiple vaccination.

ELISA, enzyme‐linked immunosorbent assay i.m., intramuscularly KLH, keyhole limpet haemocyanin OVA, ovalbumin.
Antibodies to the costimulatory molecule CD86 interfere with ultraviolet radiation‐induced immune suppression
Authors: ULLRICH S. E. , PRIDE* M. W. , MOODYCLIFFE A. M.
Published: March 2003

Although almost all of the energy contained within the ultraviolet (UV) wavelengths of solar radiation is absorbed within the epidermis and upper layers of the dermis, UV irradiation can suppress the immune response to antigens introduced at distant, non‐irradiated body sites. The production of immune modulatory cytokines, such as interleukin‐10 (IL‐10), by UV‐irradiated keratinocytes and its effect on T helper type 1 (Th1)/Th2‐cell balance are thought to play a major role in the induction of systemic immune suppression. Because it is suggested that costimulatory molecules, such as CD80 and CD86, differentially stimulate Th1 and Th2 cells we wished to investigate the role of these costimulatory molecules in the activation of immune suppression. We injected UV‐irradiated mice with monoclonal antibodies to CD80 and CD86 and asked what effect, if any, this would have on UV‐induced immune suppression. Anti‐CD86, but not anti‐CD80 or control rat IgG, blocked UV‐induced immune suppression. Moreover, monoclonal anti‐CD86 blocked the induction of suppressor T cells normally found in the spleens of the UV‐irradiated mice. Monoclonal anti‐CD86 also reversed the UV‐induced impairment of systemic antigen‐presenting cell function. IL‐10 was detectable in the serum of UV‐irradiated mice as compared with normal controls, and injecting UV‐irradiated mice with anti‐CD86, but not anti‐CD80 or control rat IgG, blocked the secretion of IL‐10 into the serum. We propose that UV exposure favours costimulation by CD86, which enhances the production of serum IL‐10, thus suppressing Th1‐cell‐mediated immune reactions.
A novel immunosuppressant, FTY720, increases the efficiency of a superantigen‐induced peripheral T‐cell deletion whilst inhibiting negative selection in the thymus
Authors: SHIMIZU  , LI  , KIMURA  , HASHIMOTO  , SUGAYA  , KUBO  , SUZUKI  , NAKAYAMA
Published: March 2003

A novel immunosuppressant, FTY720, was generated by chemical modification of ISP‐I, an immunosuppressive compound purified from culture filtrates of Isaria sinclairii. FTY720 directly induces apoptotic cell death in lymphocytes, which is believed to be the mechanism by which this drug exerts its immunosuppressive effect. We examined the effect of FTY720 treatment on antigen‐induced apoptotic cell death in peripheral T cells and thymocytes. A superantigen, staphylococcus enterotoxin B (SEB), induces T‐cell antigen receptor (TCR) Vβ‐specific apoptotic cell death in mature T cells in vivo. In this well‐documented experimental system, FTY720 administration significantly enhanced the efficiency of superantigen‐induced T‐cell deletion. We also determined that apoptotic cell death with DNA fragmentation induced in T‐hybridoma cells after stimulation in vitro with anti‐TCR antibodies was enhanced in the presence of non‐cytolytic doses of FTY720. In sharp contrast, negative selection of T cells in the thymus, another example of antigen‐induced apoptosis, was found to be inhibited by FTY720 treatment. A rescue effect was observed on clonal deletion in the H‐Y‐specific TCRαβ transgenic male thymus. In a chicken egg albumin (OVA)‐specific TCRαβ transgenic system, OVA‐induced apoptotic cell death of CD4+CD8+ thymocytes was also inhibited by FTY720 injection. Thus, FTY720 increased the susceptibility of mature T cells to TCR‐mediated apoptosis but decreased that of immature thymocytes. The results in this report suggest that the potent immunosuppressive effect of FTY720 is, in part, a result of the augmentation of effects on antigen‐induced apoptosis in mature T cells, and that two distinct apoptotic cell death pathways are operating in mature and immature T cells.
Immunodeficiency associated with anorexia nervosa is secondary and improves after refeeding
Authors: ALLENDE  , CORELL  , MANZANARES  , MADRUGA  , MARCOS  , MADROÑO  , LÓPEZ‐GOYANES  , GARCÍA‐PÉREZ  , MORENO  , RODRIGO  , SANZ  , ARNAIZ‐VILLENA*
Published: March 2003

Several studies have addressed the question of starvation effects on immune function by means of changes in lymphocyte subsets, cytokine induction or lymphocyte activation. Anorexia nervosa (AN) patients are severely malnourished and contradictory results have been obtained regarding the accompanying immunodeficiency, including its assignation as a part of the primary nervous disorder. In the present work, an extensive immunological function examination was carried out on 40 AN patients who were compared with a control group of 14 healthy girls. The AN patients were also classified according to their nutritional status (by the Body Mass Index: BMI), this being critical for a better understanding of these secondary immunodeficiency bases. Moreover, another immune system study was performed on five patients after refeeding. Lymphocyte subsets and function, cytokine induction and peripheral blood concentrations, and innate as well as humoral immunity were evaluated. Deregulation in the cytokine network, owing to the interaction of the central nervous (CNS) and immune systems, seems to be the initial immune alteration in AN immunodeficiency but it has not been disproved that the immunodeficiency is a direct consequence of the original psychiatric perturbation. Spontaneous high levels of circulating interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) have been observed; this is probably one of the causes of the anomalies found in the T‐cell subpopulations (mainly the naive CD4+CD45RA+ reduction and the cytotoxic CD8+ increase) and T‐cell activation status (mainly the down‐regulation of the CD2 and CD69 activation pathways). This finally leads to an impairment, not only in T‐cell function but also in T‐cell to B‐cell co‐operation. The AN specificity of these results is confirmed by the fact that these immune alterations improve after refeeding and when nutritional status becomes less critical, which also suggests that AN immunodeficiency is indeed secondary to malnutrition.
Involvement of protein kinase C‐δ in CD28‐triggered cytotoxicity mediated by a human leukaemic cell line YT
Authors: KOS  , BEAR
Published: March 2003

Ligation of CD28 molecules expressed on the surface of human leukaemic natural killer‐like YT cells triggers intracellular signals leading to cytolysis of target cells expressing CD80 or CD86 molecules. Known intracellular events include tyrosine phosphorylation, activation of phosphatidylinositol 3‐kinase, and protein kinase C (PKC). In this study, we report that PKC‐δ isoenzyme activity is required for CD28‐triggered cytotoxicity mediated by YT cells and we also demonstrate that one of the primary targets of bryostatin 1, a modulator of PKC activity, is PKC‐δ. Treatment of YT cells with bryostatin 1 caused degradation of PKC‐δ, but not other PKC isoenzymes, and completely blocked the cytolytic activity of YT cells. In addition, PKC‐δ‐specific antibody introduced into YT cells by electroporation inhibited partially the YT cell‐mediated cytotoxicity of B‐lymphoblastoid cell line JY. This effect was specific, since addition of anti‐PKC‐δ antibody‐blocking peptide in combination with anti‐PKC‐δ antibody to YT cells for electroporation, neutralized the effect of this antibody. These results demonstrate that YT cell cytolytic activity is dependent on PKC‐δ, which is selectively down‐regulated by bryostatin 1.
Analysis of T cells recruited during delayed‐type hypersensitivity to purified protein derivative (PPD) versus challenge with tuberculosis infection
Authors: PAIS‡  , SILVA  , SMEDEGAARD  , APPELBERG  , ANDERSEN
Published: March 2003

The delayed‐type hypersensitivity (DTH) to purified protein derivative (PPD) test has been used to infer about protective immunity to Mycobacterium tuberculosis and to diagnose tuberculosis. We showed that in memory tuberculosis‐immune mice both DTH to PPD and resistance to M. tuberculosis could be effectively elicited in the footpad and both reactions led to the accumulation of reactive T cells in the regional lymph nodes with a CD4+ phenotype and characterized by the secretion of high levels of interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) and no IL‐4. By adoptive transfer into nude mice of highly purified CD4+ T cells harvested during the recall of protective immunity it was confirmed that this population mediated both manifestations. However, the specificity of the T cells recruited during these processes were found to differ markedly; T cells involved in protection to a challenge with live tuberculosis bacilli recognized predominantly low‐mass culture filtrate antigens below 15 000 MW, while cells recruited during DTH to PPD were directed to molecular mass fractions between 15 000 and 31 000. Using single purified antigens we showed that the latter cells recognized the secreted mycobacterial protein Ag85B and the heat‐shock proteins, DnaK and GroEL. Protective T cells, in contrast, were characterized by a very high frequency of T cells directed to the ESAT‐6 peptide 1–20.
Interleukin‐1β partially alleviates cyclosporin A‐induced suppression of IgG1 isotype response to thyroglobulin in BALB/c mice in vivo
Authors: DALAI  , MIRIYALA  , KAR
Published: March 2003

Cyclosporin A (CsA) at 120 mg/kg body weight when injected subcutaneously into BALB/c mice along with thyroglobulin emulsified in incomplete Freund’s adjuvant (IFA) was found to suppress antigen‐specific IgG titre by 86%. Isotyping revealed that both IgG1 and IgG2a titres were suppressed by 87% and 57%, respectively. But under identical conditions when complete Freund’s adjuvant (CFA) was used, the suppression of antigen‐specific IgG, IgG1 and IgG2a titres was 50%, 51% and 55%, respectively. Injection of anti‐IL‐1β‐neutralizing hamster monoclonal antibodies along with thyroglobulin and CsA emulsified in CFA increased the suppression of antigen‐specific IgG titre. Under such conditions the IgG1 titre was suppressed more than the IgG2a titre. Recombinant human interleukin‐1 receptor antagonist (rhuIL‐1ra) also enhanced the suppression caused by CsA in the presence of CFA but control hamster immunoglobulin had no such effect. Recombinant human IL‐1β, when administered along with thyroglobulin and CsA emulsified in IFA, alleviated the suppression of antigen‐specific IgG titre and the IgG1 titre was alleviated more than the IgG2a titre. Under identical conditions, rhuIL‐1ra did not alleviate CsA‐induced suppression. Lymphocytes from the lymph nodes of thyroglobulin‐sensitized BALB/c mice when stimulated in vitro by thyroglobulin in the presence of CsA, secreted very little interferon‐γ (IFN‐γ) and IL‐4, but on addition of an optimal dose of rhuIL‐1β, IFN‐γ and IL‐4 secretion was partially restored.
T‐cell receptor Vβ gene expression in experimental lupus nephritis
Authors: SUTMULLER  , BAELDE  , OUELLETTE  , DE HEER  , BRUIJN
Published: March 2003

A limited T‐cell receptor (TCR) Vβ repertoire employed by autoreactive T cells may be related to the development and course of autoimmune diseases. Vβ repertoire skewing has been observed not only in man, but also in animal models of several human autoimmune diseases, such as MRL‐lpr mice, which spontaneously develop a systemic lupus erythematosus (SLE)‐like disease. Murine chronic graft‐versus‐host disease (GVHD) is an inducible model for SLE, involving direct interaction between donor T cells and recipient B cells. It is not known whether Vβ‐specific T‐cell subsets are pathogenically involved in this model. Retroviral superantigens such as Mls‐1 are known to have a profound impact on the TCR Vβ repertoire in mice. Restriction of the peripheral TCR repertoire may result from intrathymic expression of Mls‐1, which causes deletion of T cells expressing Vβ6, ‐7, ‐8.1, or ‐9. Mls‐1 incompatibility between donor and recipient can be used to determine the involvement of these TCR Vβ families in GVHD. In the present study we induced GVHD in several strain combinations to investigate TCR Vβ gene expression during GVHD, and the effect of Mls‐1 incompatibility on the TCR Vβ repertoire. TCR Vβ gene expression was determined using an RNase protection assay. Our results indicate that T cells expressing the Vβ2 or Vβ16 chain play an important pathogenetic role, while T cells bearing the Vβ1 or Vβ6 chain may be related to self‐limitation of the lupus‐like disease in this model.
Conserved T‐cell receptor class II major histocompatibility complex contact detected in a T‐lymphocyte population
Authors: FENG  , CHOU  , LIAW  , LAI
Published: March 2003

T‐cell receptor (TCR) interacts with an antigenic peptide deeply buried in the major histocompatibility complex (MHC) molecule. How class II MHC is contacted by TCR during antigen recognition remains largely elusive. Here we used a panel of I‐Ek mutants to identify two I‐Ek residues that were frequently contacted by TCR among a large pool of T cells specific for the same antigen. The restricted TCR interaction with I‐Ek was independent of the antigen peptides. We also identified a dominant heteroclitic residue on I‐Ek, β81H, in which mutation led to increased recognition of antigens in individual T‐cell clones. Moreover, both the conserved TCR–I‐Ek interaction and the heteroclitic TCR–I‐Ek recognition were detected in T lymphocytes freshly isolated from mice primed with the specific antigens. The identical TCR–I‐Ek interaction in a heterogeneous T‐cell population suggested the dominance of invariant TCR–class II MHC interaction.
Co‐existence of somatic hypermutation and gene conversion in hypervariable regions of single Igκ clones
Authors: LIU  , WOLF
Published: March 2003

In the rabbit, recent investigations have provided evidence that gene conversion leads to the generation of diversity of heavy chain rearranged VH–DH–JH genes. No data have been published on a similar mechanism for rabbit light chains. In our laboratory, we initially infected rabbits with Trypanosoma brucei, which stimulates B‐cell hyperplasia and hypergammaglobulinaemia. The heterozygous rabbits exhibited the Cκ1 b4 and b9 kappa light chain allotypes. After reverse transcription of mRNA, and cloning and sequencing of cDNA, the Vκ–Jκ–Cκ genes provided evidence for both somatic hypermutation and gene conversion. We saw that in each of the b4 and b9 kappa light chain cDNA, CDR1 and CDR3 carried both point mutation and provisional gene conversion traits. In the CDR2 region, point mutation and gene conversion inserts were observed in the b4 genes, with only gene conversion in two b9 genes. In the CDR regions, although some genes exhibited only somatic hypermutation or gene conversion, others showed linkage of both somatic hypermutation and gene conversion in the same sequence. This also marks the first time that somatic hypermutation and gene conversion in the same cloned CDR region has been observed in Vκ1 genes; however, it has been seen earlier in rabbit heavy chain VH sequences. Furthermore, the addition of several codons to the CDR3 segment by gene conversion may have provided a mechanism for length variation. In addition, we demonstrated that Jκ and framework region segments contained examples of somatic hypermutation. Confirmation of gene conversion necessitates that donor sequences be identified as providing the templated inserts. Thus after cloning two pseudogenes we found putative CDR3 donor segments for two CDR3 rearranged genes. The results offer additional mechanisms for the generation of diversity among rearranged rabbit kappa light chain genes. Whether there is a relationship or influence of gene conversion upon somatic hypermutation or vice versa is not discernable at present.
The role of neutralizing antibody and T‐helper subtypes in protection and pathogenesis of vaccinated mice following ocular HSV‐1 challenge
Authors: GHIASI  , WECHSLER  , CAI  , NESBURN  , HOFMAN
Published: March 2003

In order to determine the possible correlation of specific immune responses with protection against mortality and ocular disease following ocular herpes simplex virus type 1 (HSV‐1) challenge, BALB/c mice were vaccinated with different doses and regimens of baculovirus‐expressed gD. Neutralizing antibody, virus titres in the eyes, corneal scarring, and survival were measured. In addition, infiltration into the cornea of CD4+ T cells and cells containing the lymphokines interleukin‐2 (IL‐2), IL‐4, IL‐6 and tumour necrosis factor‐α (TNF‐α) were monitored on days 3, 7, 10, 14 and 21 post‐challenge by immunocytochemistry. The vaccination regimens used induced varying degrees of immune responses and protection upon ocular challenge with HSV‐1. Our results suggest that neutralizing antibody was the most important immune response in protecting mice against lethal ocular challenge and corneal scarring. TNF‐α and IL‐2 were not crucial in terms of survival and corneal scarring, since gD1 (one vaccination with 1 μg of gD) and gD0.1 (one vaccination with 0·1 μg of gD), both of which provided high levels of protection, showed no TNF‐α or IL‐2 expression. However, TNF‐α and IL‐2 were crucial in terms of virus clearance from the eyes, since gD3 (three vaccinations with 1 μg of gD), which had less virus in their eyes, had high numbers of TNF‐α and IL‐2 infiltrates. Finally, mock‐vaccinated mice were not protected from death and corneal disease following HSV‐1 challenge. Eyes of mock‐vaccinated mice had little or no TNF‐α or IL‐2 responses and the strongest IL‐4 and IL‐6 responses.
Characterization in vitro and in vivo of the pig analogue of human CD59 using new monoclonal antibodies
Authors: HANNA  , WILLIAMS  , VAN DEN BERG  , MORGAN
Published: March 2003

CD59 is the sole characterized regulator of the complement membrane attack complex in humans. It is very widely and abundantly distributed, being present on all circulating cells, endothelia and epithelia, and in most tissues. CD59 analogues in rodents are distributed similarly. Interest in complement regulation in the pig has developed out of the current enthusiasm to exploit this species as a donor in xenotransplantation of organs to humans. We have recently isolated and cloned the pig analogue of human CD59. We here report the development and characterization of monoclonal antibodies against pig CD59. We have used these antibodies to develop efficient methods for the purification of pig CD59 to homogeneity from erythrocyte membranes and have obtained new information on the structure and function of the purified protein. The antibodies were found to function well in immunohistochemistry and have been used to perform a comprehensive survey of the expression and distribution of pig CD59 on cells and in organs of normal pigs. Pig CD59, like human CD59, is broadly expressed but there are some striking differences in tissue distribution, notably the apparent lack of pig CD59 on circulating platelets and on a subset of leucocytes in blood and lymphoid organs. The reported findings have important implications for the current approaches to avoiding complement‐mediated hyperacute rejection in pig‐to‐human xenografts.
Autocrine regulation of interleukin‐8 by interleukin‐1α in respiratory syncytial virus‐infected pulmonary epithelial cells in vitro
Authors: PATEL  , JIANG  , NAKAJIMA  , KUNIMOTO
Published: March 2003

Respiratory epithelial cells infected with respiratory syncytial virus (RSV) produce interleukin‐8 (IL‐8); however, the mechanisms of RSV‐induced regulation of IL‐8 are poorly understood. In the present study, the regulation of IL‐8 by RSV was evaluated using pulmonary type II‐like epithelials (A549). Live purified RSV (pRSV) induced a significant increase in IL‐8 after 8 hr of exposure, while conditioned supernatants from pRSV‐infected A549 cells (cRSV) induced IL‐8 production in fresh A549 cultures within 4 hr of infection. Furthermore, cRSV that had been rendered non‐infectious by ultraviolet‐irradiation (UV‐cRSV) or ribavirin treatment also induced an increased production of IL‐8 in fresh A549 cells, suggesting that RSV induced the synthesis of a soluble mediator(s) which in turn enhanced the synthesis of IL‐8. We have previously shown that RSV‐infected A549 cells produce IL‐1α, IL‐1‐β and tumour necrosis factor‐α (TNF‐α), which by themselves are known to induce the synthesis of IL‐8. Preincubation of UV‐cRSV or simultaneous incubation of pRSV with recombinant IL‐1 receptor antagonist almost completely blocked (95–98%) the production of IL‐8 by A549 cells. Furthermore, incubation with neutralizing antibodies against IL‐1α, IL‐1β and TNF‐α showed that IL‐1α was the predominant soluble mediator that enhanced the mRNA expression and synthesis of IL‐8. IL‐1β and TNF‐α induced the synthesis of IL‐8 at 24 hr, but partially inhibited the synthesis at 48 hr. In summary, these experiments provide direct evidence for an autocrine mechanism of enhanced IL‐8 production in RSV‐infected epithelial cells that is primarily mediated by IL‐1α. In clinical settings, inhibitors of IL‐1α may be useful in suppressing inflammation due toIL‐1α as well as IL‐8.
B lymphocytes in lymph nodes and peripheral blood are important for binding immune complexes containing HIV‐1
Authors: JAKUBIK  , SAIFUDDIN  , TAKEFMAN  , SPEAR
Published: March 2003

We investigated the interaction of HIV immune complexes (HIV IC) with mononuclear cells from lymph nodes and blood. While antibody alone did not affect binding of HIV IC to mononuclear cells, antibody plus complement increased binding by as much as 10‐fold and complement alone also increased binding slightly. Most of the increased binding of HIV IC to mononuclear cells was blocked by heat‐inactivation of complement and by OKB7 monoclonal antibody, indicating that virus binding was to CR2 on B cells. A similar pattern of antibody and complement dependence for binding of HIV IC was observed with two model systems; Raji and Arent B‐cell lines. Most of the HIV IC that bound to lymph node cells were not internalized, but remained on the cell surface and were gradually released. However, even after 48 hr some HIV IC could be detected bound to cells. Under certain conditions, HIV IC were infectious for T cells if bound to B cells but not infectious if added directly to T cells. Additionally, HIV IC bound to B cells led to higher virus replication. These studies show that B lymphocytes from blood and lymph nodes can transfer infectious HIV IC to T cells.
Molecular characterization of U937‐dependent T‐cell co‐stimulation
Authors: STONEHOUSE  , WOODHEAD  , HERRIDGE  , ASHRAFIAN  , GEORGE  , CHAIN  , KATZ
Published: March 2003

U937 cells provide a co‐stimulatory signal for CD3‐mediated T‐cell activation which is independent of the CD28/CD80/CD86 interaction. This study set out to identify which molecules contribute to this co‐stimulatory activity. Monoclonal antibodies (mAb) to the known accessory molecules CD11a, CD18, CD54 and CD45, all inhibited T‐cell proliferation. Although CD11a/18 mAb inhibited U937/T‐cell cluster formation as well as proliferation, CD45 enhanced the size of the clusters formed, suggesting that this was not the only mechanism of inhibition. The alternative co‐stimulatory pathway provided by U937 cells preferentially stimulated a response in the CD18+ T‐cell population, and this reflected the reduced sensitivity of CD8+ T cells to CD28‐mediated activation. Monoclonal antibodies to three molecules, CD53, CD98 and CD147, also inhibited U937‐dependent T‐cell proliferation. The mAb to CD98 and CD147 were inhibitory when prepulsed on to the U937 cells, suggesting an effect mediated by these molecules on the antigen‐presenting cell.
U5 monoclonal antibody identifies a novel lymphocyte surface antigen preferentially expressed in human circulating natural killer cells with high cytotoxic activity
Authors: IKURA  , TERAO  , MATSUZAKI  , INOUE‐MURAYAMA  , MURAYAMA
Published: March 2003

U5 monoclonal antibody (mAb), developed against Japanese monkey lymphocytes, identified a glycoprotein of 32 000 MW that is expressed in a subset of human circulating natural killer (NK) cells. The distribution of U5 molecules was restricted among CD16+ cells, and U5 antigen was preferentially expressed in the CD38+ subset. U5+ CD16+ CD56+ cells were highly active on NK assay against K562 target cells. Variations in cytolytic activities and mRNA expression of perforin, granzyme B and Fas ligand (FasL) were observed in U5− CD16+ CD56+ cells depending on the donor. We found that in some donors, a phenotypically mature (CD16+ CD56+) but functionally immature subset was present in the peripheral circulation. The U5− CD16+ CD56+ cells of some donors exhibited negligible cytolytic activity with no detectable expression of the above mRNAs, whereas those of the other donors had a significant but lower cytolytic activity with a reduced expression of granzyme B mRNA as compared with those of U5+ CD16+ CD56+ cells. Concanavalin A (Con A) stimulation induced an expression of U5 antigen in U5− CD16+ CD56+ cells accompanied by an up‐regulation of granzyme B mRNA expression. These findings suggest that U5 antigen may be a novel molecule involved in the maturation or differentiation of human circulating NK cells.
Polyclonal expansion of TCRBV2‐ and TCRBV6‐bearing T cells in patients with Kawasaki disease
Authors: YOSHIOKA  , MATSUTANI  , IWAGAMI  , TOYOSAKI‐MAEDA  , YUTSUDO  , TSURUTA  , SUZUKI  , UEMURA  , TAKEUCHI  , KOIKE  , SUZUKI
Published: March 2003

We examined T‐cell receptor (TCR) usage, cytokine production and antibody responses to superantigens in patients with Kawasaki disease (KD) to facilitate a better understanding of the immunopathogenesis of KD. The mean percentage of VB2‐ or VB6.5‐bearing T cells in peripheral blood mononuclear cells (PBMC) of patients with acute‐phase KD was significantly higher than that of patients in the convalescent phase of KD or in healthy donors. Expansion of VB2‐ or VB6.5‐bearing T cells was polyclonal because DNA sequences in the complementarity determining region 3 of VB2‐ and VB6.5‐positive cDNA clones were all different from each other. The plasma levels of interleukin (IL)‐1β, IL‐2, IL‐6, IL‐8, IL‐10, interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and granulocyte colony‐stimulating factor (G‐CSF) were elevated in the acute phase of KD. We previously reported that streptococcal pyrogenic exotoxin C (SPEC) was a potent stimulator of VB2‐ and VB6.5‐positive T cells and, furthermore, serum levels of anti‐SPEC antibodies were significantly higher in patients with acute and convalescent KD than in age‐matched controls. The results of the present study, together with those of our previous report, suggest that SPEC induces activation and polyclonal expansion of VB2‐ and VB6.5‐positive T cells, and that SPEC‐induced activation of T cells may lead to the pathogenesis of KD.
A mutational analysis of the Aβz/Aαd major histocompatibility complex class II molecule that restricts autoreactive T cells in (NZB×NZW)F1 mice. The critical influence of alanine at position 69 in the Aαd chain
Authors: SAI  , MINE  , FUKUOKA  , KOARADA  , KIMOTO
Published: March 2003

Autoimmune symptoms of (NZB×NZW)F1 (H‐2d/z) mice are reported to be critically related to the heterozygosity at the H‐2 complex of the murine major histocompatibility complex (MHC). We previously showed that several Aβz/Aαd MHC class II molecule‐restricted autoreactive T‐cell clones from B/WF1 mice were pathogenic upon transfer to preautoimmune B/WF1 mice. In this study, to identify the crucial amino acid residues in Aβz/Aαd molecules for T‐cell activation, we generated a panel of transfectant cell lines. These transfectant cell lines express the Aβz/Aαd MHC molecules with a mutation at each residue α11, α28, α57, α69, α70, α76 of Aαd chain and β86 of Aβz chain. Replacing α69 alanine with threonine, valine or serine completely eliminated the ability to stimulate autoreactive T‐cell clones without affecting the ability to present foreign antigen keyhole limpet haemocyanin (KLH) or l‐plastin peptide to specific T‐cell clones. Replacing β86 valine with aspartic acid resulted in a decrease in the stimulation for antigen‐reactive as well as autoreactive T‐cell clones. Substitutions at other residues had minimal or no effect on the stimulation of either auto‐ or antigen‐reactive T‐cell clones. These results suggest that alanine at residue 69 of the Aαd chain is critical for the activation of autoreactive Aβz/Aαd‐restricted T‐cell clones. Possible explanations for this are discussed.
Expression of CD1D mRNA transcripts in human choriocarcinoma cell lines and placentally derived trophoblast cells
Authors: JENKINSON  , WAINWRIGHT  , SIMPSON  , PERRY  , FOTIADOU  , HOLMES
Published: March 2003

Human placental trophoblast is critically involved in mediating maternal tolerance of the fetal semiallograft. Genes encoding highly polymorphic major histocompatibility complex (MHC) class I and class II antigens that could provoke maternal immune rejection responses are silenced in trophoblast. However, several MHC class I or class I‐related products exhibiting reduced or negligible polymorphism are expressed and assumed to be functionally involved in maintaining pregnancy. The CD1 gene family encodes non‐polymorphic MHC class I‐like products that have the unusual ability to present non‐peptide antigens to T cells. One member, CD1D, is expressed in certain epithelial cells and interacts with a specific T‐cell subset that may promote the development of Th2‐mediated responses believed to be associated with pregnancy. In this study we examined the expression of CD1D in human trophoblast cell lines and placentally derived trophoblast cells by reverse transcriptase–polymerase chain reaction using CD1D‐specific oligonucleotide primers. We have found that CD1D mRNA transcripts are expressed in trophoblast cells and cell lines. We have also identified a novel alternatively spliced CD1D mRNA transcript lacking exon 4. Exon 4‐intact and exon 4‐deficient CD1D transcripts appear to be differentially expressed in different trophoblast and non‐trophoblast cell populations. Our studies suggest that at least one member of the CD1 family is transcribed in human trophoblast.
Secretion of parasite‐specific immunoglobulin G by purified blood B lymphocytes from immune individuals after in vitro stimulation with recombinant Plasmodium falciparum merozoite surface protein‐119 antigen
Authors: Garraud  , Diouf  , Holm  , Nguer  , Spiegel  , Perraut  , Longacre
Published: March 2003

The C‐terminal 19 000 MW fragment of merozoite surface protein‐1 (MSP119) is one of the most promising candidate antigens for a malaria vaccine. Baculovirus recombinant Plasmodium falciparum MSP119 has been used to define conditions for the in vitro production of specific antibodies by purified human blood B cells in a culture system where T‐cell signals were provided by the engagement of CD40 molecules and exogenous cytokines. MSP119 preferentially induced surface immunoglobulin G (IgG) ‐positive (sγ+) B lymphocytes from P. falciparum‐immune donors to differentiate and produce antigen‐specific IgG. In contrast, naïve B cells or cells from non‐immune donors could not be induced to secrete parasite‐specific IgG in vitro. Although IgG secretion was obtained in the absence of exogenous cytokines, it was dependent on B‐cell‐derived interleukin‐10 (IL‐10) and/or B‐cell factor(s) under the control of IL‐10, since IgG levels were significantly decreased in the presence of neutralizing anti‐IL‐10 antibodies. These results demonstrate at the cellular level that a single malaria vaccine candidate polypeptide can direct parasite‐specific antibody production mediated by the secretion of potentiating factors.
Do self‐perpetuating B lymphocytes drive human autoimmune disease?
Authors: Edwards  , Cambridge  , Abrahams
Published: March 2003

Normal immunological memory is thought to be underpinned by T lymphocytes. However, in rheumatoid arthritis there are indications that T‐lymphocyte control has been subverted by self‐perpetuating B lymphocytes. Potential mechanisms in other autoimmune states are less clear, but a number of observations suggest that misappropriation of immunological memory by B lymphocytes may be a common feature of human autoantibody‐associated disease. Put simply, autoantibodies drive their own production. If so, the availability of safe B‐lymphocyte‐depleting agents provides a potential means for reversal of autoimmunity.
Interleukin‐10 activates heat‐shock protein 90β gene expression
Authors: Ripley  , Stephanou  , Isenberg  , Latchman
Published: March 2003

Elevated levels of the cytokine interleukin‐10 (IL‐10) have been reported in patients with active systemic lupus erythematosus (SLE). Any role for IL‐10 in the pathogenesis of SLE is likely to involve the activation of expression of specific genes within its target cells. We have previously reported elevated levels of the 90 000 MW heat‐shock protein (hsp 90) and autoantibodies to hsp 90 in patients with SLE. Recent studies have shown that the cytokine IL‐6 activates hsp 90 gene expression via specific transcription factors that include STAT‐3 (signal transducer and activator of transcription 3). In view of the known role of STAT proteins in IL‐10 signalling pathways, we have investigated the effect of IL‐10 on hsp 90 gene expression. Here we report that IL‐10 enhances the expression of hsp 90 in both a human hepatoma cell line (HepG2) stably expressing the human IL‐10 receptor and peripheral blood mononuclear cells (PBMC). In reporter gene assays IL‐10 is able to activate both the hsp 90α and hsp 90β promoters directly. Furthermore, a short region of the hsp 90β promoter which is activated in response to IL‐10, contains a STAT‐3 binding site. This element but not a mutant derivative unable to bind STAT‐3, is able to confer a response to IL‐10 on a heterologous promoter. These results may be understood in terms of the shared signalling mechanisms of IL‐10 and IL‐6 and provide evidence of a role for IL‐10 in the overexpression of hsp 90 in SLE, with possible pathological consequences.
Enhanced antigen‐presenting activity and tumour necrosis factor‐α‐independent activation of dendritic cells following treatment with Mycobacterium bovis bacillus Calmette–Guérin
Authors: Kim  , Lee  , Kim  , Park  , Choe  , Choe  , Kim  , Lee E. , Lim
Published: March 2003

Dendritic cells (DCs) are most potent among the antigen‐presenting cells and are believed to be crucial for the initiation of a primary T‐cell response to foreign antigens. Mycobacterial infection within macrophages is controlled by cell‐mediated immunity. To elucidate the stimulation of immune response by Mycobacterium bovis bacillus Calmette–Guérin (BCG), we purified DCs from precursor cells in human peripheral blood mononuclear cells (PBMC) by culturing them with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4) and characterized their surface antigen expression. The interaction of cultured DCs with BCG resulted in increased surface expression of several DC‐related marker antigens. BCG also induced reduction of endocytosis, enhancement of CD83 expression as well as B7 costimulatory molecules and IL‐12 production, suggesting that BCG treatment directly induces DCs to mature. BCG‐treated DCs were much more potent antigen‐presenting cells in allogeneic immune response than untreated DCs. Moreover, while the neutralization of tumour necrosis factor‐α (TNF‐α) significantly blocked the DC maturation induced by lipopolysaccharide (LPS), it could not inhibit the induction of DC maturation by the BCG treatment, indicating that TNF‐α production plays a minor role in the BCG‐induced DC maturation. However, the neutralization of TNF‐α resulted in decreased IL‐12 production by activated DCs. These results suggest that infection with BCG might evoke direct activation and maturation of DC and the general immune stimulant effect of BCG might be related with the activation of DCs.
Rheumatoid factor isotype switch and somatic mutation variants within rheumatoid arthritis synovium
Authors: Williams  , Moyes  , Mageed
Published: March 2003

The presence of clonally‐related B‐lymphocyte aggregates within synovial lining tisue of rheumatoid arthritis (RA) patients suggests a germinal centre‐like reaction, which may hold implications for disease pathogenesis and the causes of chronic inflammation. We studied 250 rheumatoid factor (RF) heavy‐chain sequences cloned from the synovium of three patients with RA, to determine whether they undergo both somatic mutation and isotype switching consistent with this hypothesis. Size analysis of immunoglobulin heavy‐chain cDNAs from synovial RF+ B cells revealed oligoclonal RF+ populations and identically‐sized VH‐D‐JH transcripts of different immunoglobulin isotypes. Sequencing of individual inserts selected from cloned immunoglobulin heavy‐chain cDNAs demonstrated a clonal relationship between immunoglobulin M (IgM) RF and IgA RF, suggesting that this isotype switch occurred in synovium. Furthermore, most somatic mutations were found to have occurred after this isotype switch. This finding suggests that the RA synovial microenvironment sustains somatic mutation and isotype switching in RF‐specific B lymphocytes akin to secondary lymphoid organs.
Nitric oxide selectively decreases interferon‐γ expression by activated human T lymphocytes via a cGMP‐independent mechanism
Authors: Roozendaal R. , Vellenga E. , Postma D. S. , De Monchy J. G. R. , Kauffman H. F.
Published: March 2003

The role of exogenous nitric oxide (NO) on the expression of interleukin (IL)‐2, IL‐4, IL‐5 and interferon‐γ (IFN‐γ) by freshly isolated human T lymphocytes was investigated. The presence of NO, generated from any of the NO‐donor compounds, S‐nitroso‐N‐acetyl‐d,
l‐penicillamine (SNAP), DPTA‐nonoate (DPTA) or DETA‐nonoate (DETA), added 15 min prior to T‐cell stimulation (for 24 hr) with anti‐CD3/anti‐CD28 monoclonal antibodies (mAbs), resulted in up to 50% inhibition of IL‐4, IL‐5 and IFN‐γ secretion. In contrast, IL‐2 secretion was not inhibited. Using the guanylate cyclase inhibitor, LY83583, it was shown that the inhibition of IL‐4 and IL‐5 was cGMP dependent, whereas additional mechanisms mediated the inhibition of IFN‐γ. Exposure of T cells to the NO‐donor compounds for 24 hr prior to stimulation resulted in a more pronounced inhibition of IFN‐γ secretion by DPTA and DETA (P < 0·01), despite the fact that NO generation could no longer be detected. Under these conditions, IL‐4 secretion was not inhibited and IL‐5 secretion was inhibited to a lesser extent (P < 0·01 for SNAP and DPTA, P > 0·05 for DETA). IL‐2 secretion was inhibited after 24 hr of preincubation with the NO‐donor compounds, whereas it was not directly affected by NO. The increased inhibitory effects on IFN‐γ and IL‐2 secretion could not be accounted for by the antiproliferative effects of the NO‐donor compounds, which were diminished after 24 hr of preincubation relative to 15 min of preincubation. For IFN‐γ, the inhibition was at least partially effected at the transcriptional level as shown by decreased mRNA accumulation. These data show that NO can modulate the balance between the expression, by human T‐lymphocytes, of T helper 1‐ and T helper 2‐type cytokines, through selective and persistent inhibition of the expression of IFN‐γ via a cGMP‐independent mechanism.
Regulation of B‐1 cell activation and its autoantibody production by Lyn kinase‐regulated signallings
Authors: Ochi H. , Takeshita H. , Suda T. , Nisitani S. , Honjo T. , Watanabe T.
Published: March 2003

The src‐family protein tyrosine kinase, Lyn, has been reported to play a crucial role in the regulation of B‐cell antigen receptor (BCR)‐mediated signalling. To elucidate the role of Lyn in the maintenance of immunological tolerance and the prevention of B‐1 cell activation and its autoantibody production, Lyn‐deficient mice were crossed with transgenic mice carrying the immunoglobulin heavy and light chain genes encoding an autoantibody against mouse red blood cells. In the transgenic mice, most peripheral B cells expressed the B‐1 cell phenotype. When the transgenic mice were bred in specific pathogen‐free (SPF) conditions, B‐1 cells were anergic and did not produce any autoantibody. In contrast, Lyn‐deficient transgenic mice kept in the same SPF conditions revealed markedly increased numbers of activated B‐1 cells and developed severe autoimmune haemolytic anaemia. Moreover, the mice had a huge splenomegaly containing a remarkable accumulation of erythroblasts, resulted from extramedullary erythropoiesis, in addition to the increased numbers of lymphoblast‐like cells of the B‐1 cell lineages. The present study demonstrates a crucial role of Lyn kinase in the regulation of B‐1 cell activation and maintenance of tolerance.