bluuebunny/crossref_metadata_2025 huggingface dataset:
Desialylation of T lymphocytes overcomes the monocyte dependency of pokeweed mitogen‐induced T‐cell activation
Authors: GALLART T. , ANGEL DE LA FUENTE M. , JOSEP BARCELÓ J. , ALBEROLA‐ILA J.
Published: November 2003

Activation of T lymphocytes by pokeweed mitogen (PWM) is strictly monocyte (Mo)‐dependent and results in T‐cell mitogenesis and interleukin‐2 (IL‐2) secretion, coupled with an inability to utilize IL‐2 due to an impaired expression of functional IL‐2 receptor (IL‐2R). Such IL‐2R impairment could arise in PWM‐activated T cells themselves or, alternatively, be the result of Mo‐derived influences, as it is known that PWM binds Mo strongly and does not or poorly binds lymphocytes, and Mo becomes rapidly destroyed in PWM‐stimulated cultures of blood mononuclear cells or T cells plus Mo. The present study investigated these possibilities. The results show for the first time that desialylation of T lymphocytes strongly increases their PWM‐binding capacity and, in addition, overcomes the Mo requirement for PWM to induce T‐cell mitogenesis and IL‐2 secretion. Such secreted IL‐2 levels were even higher that those found in cultures of Mo‐dependent PWM‐activated T lymphocytes but, similarly to the latter, PWM‐activated desialylated purified T lymphocytes exhibited negligible high‐affinity IL‐2 binding capacity and an inability to utilize the IL‐2 they produced. These effects were not due to desialylation itself, as indicated by data obtained with peanut agglutinin, a lectin that becomes strongly reactive with desialylated T lymphocytes. The data clearly indicate the existence of PWM‐related events capable of impairing the expression of functional IL‐2R without affecting IL‐2 secretion, and indicate that such events are due to mechanisms arising at the level of PWM‐activated T cells themselves.
Delayed‐type hypersensitivity elicited by synthetic peptides complexed with Mycobacterium tuberculosis hsp 70
Authors: ROMAN E. , MORENO C.
Published: November 2003

Four synthetic peptides bearing dominant CD4+ T‐cell epitopes of the 19 000 and 38 000 MW proteins of Mycobacterium tuberculosis were used to provoke a delayed‐type hypersensitivity (DTH) reaction in mice previously immunized with recombinant 19 000 and 38 000 MW proteins. It was found that an effective enhancement of the DTH reaction was obtained if the peptides were administered as a complex with M. tuberculosis hsp 70 protein. The increase in reactivity was not obtained when hsp 70 and peptide were co‐injected at the same site, but not in complex, or when the specific peptide was displaced from the complex by an irrelevant peptide with high capacity to bind hsp 70. One of the antigenic peptides whose capacity to complex with hsp 70 is low, failed to show the enhancement of DTH when injected together with hsp 70.
Kinetics of cellular infiltration and cytokine production during the efferent phase of a delayed‐type hypersensitivity reaction
Authors: BUCHANAN K. L. , MURPHY J. W.
Published: November 2003

Cell‐mediated immunity is a primary host resistance mechanism against many infectious organisms and is responsible for leucocyte recruitment to the infection site. Delayed‐type hypersensitivity (DTH) reactions are in vivo correlates of cell‐mediated immunity and have long been used to assess the level of cell‐mediated immune (CMI) responsiveness to specific antigens. It has been difficult to study the kinetics of cellular influx and cytokine composition at the site of an on‐going CMI reaction. Consequently, knowledge of the sequential events occurring during the efferent phase of a CMI response is incomplete. Here we report on the use of a gelatin sponge model for evaluating the progression of events during the effector phase of a DTH reaction to antigens of the mycotic organism Cryptococcus neoformans. Previously, we have shown that 24 hr after antigen injection into sponges in infected or immune mice, the leucocyte types infiltrating the sponges are consistent with a classical murine DTH reaction. Through kinetic studies, we show here that neutrophils are the first leucocytes to appear in DTH‐reactive sponges, followed by increases in lymphocyte and then monocyte numbers. Tumour necrosis factor (TNF), interleukin‐2 (IL‐2), interferon‐γ (IFN‐γ) and IL‐5 were elevated in DTH‐reactive sponges compared with control sponges, and each cytokine had a relatively unique temporal profile. IL‐4 was not detectable in the sponges. Together our data indicate that the expression of a CMI response comprises a well‐regulated sequential influx of leucocytes that contribute to the lymphokine composition of the reaction.
Pretreatment of lymphocytes with mercury in vitro induces a response in T cells from genetically determined low‐responders and a shift of the interleukin profile
Authors: HU H. , ABEDI‐VALUGERDI M. , MÖLLER G.
Published: November 2003

Mercury can induce autoimmune disease in susceptible mouse strains. We found that in vitro mercuric chloride induced a high proliferative response in spleen lymphocytes from mercury‐susceptible SJL mice, but a low response in resistant mice, such as C57BL/6 (H‐2b), A/J (H‐2a) and CBA (H‐2k) mice. However, a high proliferative response was obtained with lymphocytes from all tested low‐responder mice by pretreating them in vitro for 1–3 days with mercuric chloride and then wash away the excess mercury. Both CD4+ and CD8+ T cells were activated in the restored response, but CD4+ T cells was the major responding cell population, as in high‐responder mice. We also measured the cytokine production at the protein level after mercury stimulation in vitro. We found that in mercury stimulation the different culture conditions resulted in different patterns of cytokine production. The continuous presence of mercury induced interleukin‐2 (IL‐2) and interferon‐γ, but not IL‐4 production in spleen cells from both high‐ and low‐responder mice. In contrast, by pretreating the cells with mercury and then washing, spleen cells from both high and low‐responder mice produced IL‐4. Our results suggest that spleen cells from both mercury‐susceptible and ‐resistant mice have the potential to respond to mercury in vitro and produce both Th1‐ and Th2‐type cytokines. But the mercury‐induced cytokine profile can shift depending on the conditions for activation.
Intercellular adhesion molecule‐1 on eosinophils is involved in eosinophil protein X release induced by cytokines
Authors: HORIE S. , OKUBO Y. , HOSSAIN M. , MOMOSE T. , SUZUKI J. , ISOBE M. , SEKIGUCHI M.
Published: November 2003

Recent evidence suggests that adhesion molecules play important roles in eosinophil functions such as degranulation and superoxide anion production. CD11b/CD18 (Mac‐1) and CD49d/CD29 (VLA‐4) are involved in eosinophil–endothelial adhesion through their counterligands, intercellular adhesion molecule‐1 (ICAM‐1; CD54) and vascular cell adhesion molecule‐1 (VCAM‐1), respectively. CD54 is also induced on eosinophils by cytokine stimulation. We hypothesized that CD54 on human eosinophils may participate in eosinophil degranulation. CD54 was induced on eosinophils by a combination of human recombinant granulocyte–macrophage colony‐stimulating factor (rGM‐CSF) and human recombinant tumour necrosis factor‐α (rTNF‐α) within 2 hr of incubation, as determined by flow cytometric analysis. Recombinant GM‐CSF alone induced a slight but significant CD54 expression on eosinophils. Release of eosinophil protein X, an indicator of eosinophil degranulation, was induced by rGM‐CSF and this effect was synergistically enhanced by adding rTNF‐α. To determine the role of newly expressed CD54 in eosinophil degranulation, a blocking assay was performed using monoclonal antibody (mAb) against CD54 and CD18. Anti‐CD18 mAb and anti‐CD54 mAb markedly inhibited eosinophil degranulation induced by rGM‐CSF or a combination of rGM‐CSF and rTNF‐α. On the other hand, anti‐CD54 mAb had little effect on rGM‐CSF‐ or rGM‐CSF/rTNF‐α‐induced adhesion of eosinophils, whereas anti‐CD18 mAb significantly inhibited eosinophil adhesion. These results indicate that CD54 on eosinophils plays an important role in the eosinophil degranulation and that eosinophils are capable of interacting with other β2 integrin‐positive cells.
The antigen receptors on mature and immature T lymphocytes are coupled to phosphatidylcholine‐specific phospholipase D activation
Authors: REID P. A. , GARDNER * S. D. , WILLIAMS D. M. , HARNETT† M. M.
Published: November 2003

Phosphatidylcholine–phospholipase D has been proposed to play a key role in the transduction of the proliferative responses of a wide range of mitogens and growth factors. We now report that the antigen receptors on T lymphocytes derived from human tonsillar or murine splenic preparations are coupled to phosphatidylcholine (PtdCho)–phospholipase D (PLD) activation following stimulation of these T cells with anti‐CD3 antibodies. However, since we also demonstrate that the antigen receptors on murine thymocytes are coupled to PtdCho–PLD activation, we propose that it is unlikely that this PLD pathway plays a central role in the transduction of T‐cell proliferative responses, but rather, may be involved in either driving cells into cycle or maintaining cell cycle progression, processes required both for proliferation and activation‐induced cell death. Whilst the molecular mechanisms underlying T‐cell receptor (TCR)‐coupling to PtdCho–PLD activation in these cells have not been fully defined, kinetics studies and experiments using pharmacological inhibitors of protein tyrosine phosphatases (PTPases) and reconstituting CD3‐coupled PtdCho–PLD activity in streptolysin‐O permeabilized cells, suggest that the TCR/CD3 complex, under optimal conditions of activation, may be predominantly coupled to PtdCho–PLD activation downstream of tyrosine phosphorylation of phospholipase C‐γ (PLC‐γ), phosphatidylinositol (PtdIns)P2hydrolysis, calcium mobilization and protein kinase C (PKC) activation.
Mutations affecting the superantigen activity of staphylococcal enterotoxin B
Authors: BRIGGS C. , GARCIA C. , ZHANG L. , GUAN L. , GABRIEL J. L. , ROGERS T. J.
Published: November 2003

As a superantigen, staphylococcal enterotoxin B (SEB) possesses the ability to bind to major histocompatibility complex class II molecules and be recognized by T cells bearing certain T‐cell receptor (TCR) Vβ alleles. Other investigators have utilized site‐specific mutagenesis to generate amino acid substitutions to identify residues that may be involved in the interaction with the TCR β‐chain. In an attempt further to define the face of the SEB molecule involved in the interaction with the β‐chain, we have employed a polymerase chain reaction (PCR)‐based, site‐specific mutagenesis method to generate amino acid substitutions with altered superantigen activity. Our results show that valine at position 169 appears to be involved in the function of this superantigen, since each of several substitutions at this position exhibit a significantly reduced ability to induce T‐cell proliferation. Analysis of the responding T cells to the residue 169 substitution shows that the mutant toxins maintain TCR Vβ selectivity. At the same time, mutation of the proximal histidine at position 166 does not alter the superantigen activity of SEB. Radiolabelled binding analysis of these H166 and V169 mutants shows that class II‐binding activity is not significantly altered. When viewed in the context of other results reported in the literature, combined with the crystal structure of the toxin, our results suggest that the interaction with the TCR probably involves SEB residues which ring a cavity along one side of the toxin molecule.
Contrasting immunopathogenic properties of highly homologous peptides‘qc from rat and human thyroglobulin
Authors: RAO V. P. , CARAYANNIOTIS G.
Published: November 2003

The current lack of amino acid sequence data for mouse thyroglobulin (Tg) necessitates mapping of pathogenic T‐cell epitopes on heterologous Tg in mouse experimental autoimmune thyroiditis (EAT). A prevailing assumption has been that epitopes sharing a high degree of amino acid homology among heterologous Tg are likely to exhibit the same immunopathogenic properties in the same host. In this report, we have examined this concept while working with the 18‐mer rat(r)Tg(2695–13) peptide that was previously shown to elicit As‐restricted T cells and EAT in SJL mice. A major immunopathogenic T‐cell epitope was localized within the 12‐mer rTg(2695–06). It was found that the human 12‐mer homologue that carries two Ser substitutions at Glu2703 and Thr2704 exhibited contrasting properties: it failed to activate Th1 cells in lymphokine and proliferation assays; it did not cross‐react with rTg(2695–06) at the T‐cell level; and it induced only focal thyroiditis following adoptive transfer of specific lymph node cells. These data highlight the caveat involved in extrapolating results of pathogenic T‐cell epitope mapping across heterologous Tgs, even when such epitopes share a high degree of amino acid homology.
The serum resistance of malaria‐infected erythrocytes
Authors: KAWAMOTO Y. , KOJIMA K. , HITSUMOTO Y. , OKADA H. , HOLERS V. MICHAEL , MIYAMA A.
Published: November 2003

IgG and IgM antibodies were detected on non‐parasitized as well as parasitized erythrocytes (E) from mice surviving over 15 days after infection with rodent malaria, Plasmodium berghei, whereas C3 was detected exclusively on parasitized E. Parasitized E, however, were quite resistant to the haemolytic activity of guinea pig complement and effectively inactivated human C3b to iC3b on their surface. Similarly, parasitized E were extremely resistant to homologous complement as assessed by haemolysis and C3 binding even when regulatory proteins (decay‐accelerating factor, DAF; complement receptor related gene y, Crry; heat‐stable antigen, HSA) were blocked with specific antibodies. DAF and Crry were equally expressed on both normal E and parasitized E from mice within a week post‐infection; therefore, molecules that inhibit the haemolysis or C3 binding of parasitized E appear to be independent of DAF and Crry. Unexpectedly, the molecular forms of HSA and DAF in parasitized erythrocyte membranes were found to be different from those of normal erythrocyte membranes: DAF was detected as three bands (85 000, 64 000 and 30 000 MW) by immunoblotting. HSA was detected as more highly glycosylated forms than normal HSA. These alterations of DAF and HSA could be explained by the modification of membrane proteins and polysaccharides induced by parasitization, and we hypothesize that these changes of membranes or membrane proteins are involved in the resistance of parasitized E against homologous complement.
IL‐13 production by allergen‐stimulated T cells is increased in allergic disease and associated with IL‐5 but not IFN‐γ expression
Authors: TILL S. , DURHAM S. , DICKASON R. , HUSTON D. , BUNGRE J. , WALKER S. , ROBINSON D. , KAY A. B. , CORRIGAN C.
Published: November 2003

Interleukin‐13 (IL‐13) shares many, but not all, of the properties of the prototypic T‐helper type 2 (Th2) cytokine IL‐4, but its role in allergen‐driven T‐cell responses remains poorly defined. We hypothesized that allergen stimulation of peripheral blood T cells from patients with atopic disease compared with non‐atopic controls results in elevated IL‐13 synthesis in the context of a ‘Th2‐type’ pattern. Freshly isolated peripheral blood mononuclear cells (PBMC) obtained from sensitized atopic patients with allergic disease, and non‐atopic control subjects, were cultured with the allergens Phleum pratense (Timothy grass pollen) or Dermatophagoides pteronyssinus (house dust mite) and the non‐allergenic recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Supernatant concentrations of IL‐13, along with IL‐5 and interferon‐γ (IFN‐γ) (Th2‐ and Th1‐type cytokines, respectively) were determined by enzyme‐linked immunosorbent assay (ELISA). Allergen‐induced IL‐13 and IL‐5 production by T cells from patients with allergic disease was markedly elevated (P=0·0075 and P=0·0004, respectively) compared with non‐atopic controls, whereas IFN‐γ production was not significantly different. In contrast to allergen, the prototypic Th1‐type antigen M. tuberculosis PPD induced an excess of IFN‐γ over IL‐13 and IL‐5 production, and absolute concentrations of cytokines were not affected by the presence or absence of atopic disease. Addition of exogenous recombinant IFN‐γ or IL‐12, cytokines known to inhibit Th2‐type responses, significantly inhibited allergen‐driven production of both IL‐13 and IL‐5, but not T‐cell proliferation, whereas exogenous IL‐4 did not significantly affect production of IL‐13 or IL‐5. We conclude that allergen‐specific T cells from atopic subjects secrete elevated quantities of IL‐13 compared with non‐atopic controls, in the context of a Th2‐type pattern of cytokine production.
Importance of combined treatment with IL‐10 and IL‐4, but not IL‐13, for inhibition of monocyte release of the Ca2+‐binding protein MRP8/14
Authors: LÜGERING N. , KUCHARZIK T. , LÜGERING A. , WINDE G. , SORG C. , DOMSCHKE W. , STOLL R.
Published: November 2003

Expression of the two myeloic related proteins MRP8 and MRP14 is restricted to distinct stages of monocytic differentiation. Heterodimeric MRP8/14 complexes (27E10 antigen) have been shown to represent their biologically active forms. In this study, we investigated the effects of Th2‐cytokines on release of these proteins from freshly obtained blood monocytes and monocytes cultured for 7 days in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Monocytes were stimulated with pokeweed mitogen (PWM) in the presence or absence of interleukin‐13 (IL‐13), IL‐4 and IL‐10, and secretion of MRP8, MRP14 and MRP8/14 was assessed by using a sandwich enzyme‐linked immunosorbent assay system. Peripheral monocytes secreted significantly increased amounts of MRP14 and MRP8/14 but not MRP8 under stimulation with PWM. IL‐10 and IL‐4, but not IL‐13, down‐regulated the PWM‐stimulated MRP8/14 secretion in a dose‐dependent manner. Maximal inhibition required that IL‐10 and IL‐4 be added up to 1 h before or simultaneous with PWM. A combination of IL‐10 and IL‐4 even at suboptimal concentrations significantly suppressed protein secretion much more than using IL‐10 or IL‐4 at a doubled concentration alone. Peripheral monocytes cultured for 7 days in the presence of GM‐CSF showed two‐ to threefold higher protein levels compared with freshly obtained blood monocytes but responded inefficiently to either IL‐4, IL‐13, or IL‐10 alone. However, treatment with IL‐10 in combination with IL‐4 but not IL‐13 strongly suppressed MRP14 and MRP8/14 release by these cells. The unresponsiveness of 7‐day‐cultured blood macrophages suggests that more differentiated and activated cells may lose their ability to respond to anti‐inflammatory cytokines. Combined cytokine treatment may therefore more effectively control the progression of chronic inflammatory processes.
Characterization of the bovine Cα gene
Authors: BROWN W. R. , RABBANI H. , BUTLER J. E. , HAMMARSTRÖM L.
Published: November 2003

The complete genomic sequence of a bovine Cα gene is reported here. The genomic sequence was obtained from a Cα phage clone that had been cloned from a genomic EMBL4 phage vector library. The Cα sequence had previously been expressed as a chimeric antibody and identified as IgA using IgA‐specific antibodies. Intron/exon boundaries were determined by comparison of the genomic sequence with an expressed bovine Cα sequence obtained from spleen by reverse transcription‐polymerase chain reaction (RT‐PCR). Analysis of 50 Swedish bovine genomic DNA samples using genomic blots and five different restriction enzymes failed to detect evidence of polymorphism. However, PstI digests of Brown Swiss DNA showed a restriction fragment length polymorphism (RFLP), suggesting that at least two allelic variants of bovine IgA exist. Comparison of the deduced amino acid sequence of bovine IgA with sequences available for other species indicated that the highest homology was with that of swine, another artiodactyl. This was the highest homology observed for all mammalian IgA compared except for that between IgA1 and IgA2 in humans. Bovine IgA shares with rabbit IgA3 and IgA4, an additional N‐linked glycosylation site at position 282. However, the collective data indicate that cattle are like swine and rodents and unlike rabbits in having a single locus of the gene encoding IgA of this species.
Impaired calcium mobilization and differential tyrosine phosphorylation in intestinal intraepithelial lymphocytes
Authors: SANCHEZ‐GARCIA F. J. , ALLER W. W. , MCCORMACK W. T.
Published: November 2003

Intestinal intraepithelial lymphocytes (iIEL) exhibit a unique activation state characterized by the expression of activation markers and effector functions, but a minimal response to mitogenic signals in vitro. To further characterize this activation status, iIEL were compared with splenic T cells for two key activation signals, calcium mobilization and tyrosine phosphorylation. Calcium mobilization was impaired in iIEL treated with the calcium ionophores ionomycin or A23187, thapsigargin, or by CD3‐cross‐linking. The calcium mobilization defect is shared by mature and embryonic iIEL. Anti‐phosphotyrosine Western blot analysis revealed that the iIEL are able to respond to T‐cell receptor (TCR)‐mediated signals by tyrosine phosphorylation, although the patterns of phosphorylation differ from those seen in splenic T cells. We conclude that iIEL are unable to mobilize calcium in vitro, which may be due to modulation of TCR‐mediated signal transduction pathways by the microenvironment of the intestinal epithelium and/or caused by the standard isolation procedure used to prepare iIEL, which must be considered in future in vitro studies of iIEL function.
Interleukin‐7 in the skin of Schistosoma mansoni‐infected mice is associated with a decrease in interferon‐γ production and leads to an aggravation of the disease
Authors: WOLOWCZUK I. , DELACRE M. , ROYE O. , GIANNINI S. L. , AURIAULT C.
Published: November 2003

The effect of recombinant interleukin‐7 (rIL‐7) on the course of murine schistosomiasis and the development of the accompanying immune response were investigated. We demonstrated that IL‐7 expression could be detected in the skin of infected mice from 1 to 21 days following infection. We here report that intradermal injection of exogenous human IL‐7, prior to the penetration of the parasite into the skin, leads to a more severe liver pathology and an increased number of surviving adult parasites. In addition, injection of rIL‐7 alters parasite migration (estimation of burdens of young larvae in lungs and liver). Administration of rIL‐7 led to a decrease of IL‐12 and interferon‐γ‐ (IFN‐γ) specific messengers RNA in skin and, more markedly, in skin‐draining lymph nodes. The number of B220 expressing cells was increased, and T‐cell number was reduced, in IL‐7‐treated infected mice. In addition, levels of IFN‐γ and IL‐4 in sera were significantly reduced, whereas there was a shift from a Th1 to a Th2 type associated humoral response towards the egg antigens. Our experimental observations illustrate that the exogenous administration of rIL‐7 affects both the development of the host’s immune response and the behaviour of the parasite within the infected host. The early and specific production of IL‐7 in the host skin, following infection with Schistosoma mansoni, raises fascinating questions concerning the relationships between the parasite and its host at the very beginning of their interaction.
Granulocyte–macrophage colony‐stimulating factor elevates invariant chain expression in immature myelomonocytic cell lines
Authors: KLAGGE I. , KOPP U. , KOCH N.
Published: November 2003

Invariant chain (Ii) plays an important role in major histocompatibility complex (MHC) class II antigen processing and presentation and is constitutively synthesized in B lymphocytes, in macrophages, dendritic cells and in some epithelial cells. It has been shown that interferon‐γ, tumour necrosis factor‐α and interleukin‐4 co‐regulate Ii and MHC class II expression in various cell types. We describe here a novel regulation of Ii expression in macrophages. Treatment of the premature monocytic cell lines WEHI 265.1, M1 and WEHI‐3B with recombinant granulocyte–macrophage colony‐stimulating factor (GM‐CSF) strongly enhances Ii expression while class II expression is not induced. In contrast, GM‐CSF did not enhance Ii in mature macrophage cell lines. The increase of Ii expression in WEHI 265.1 cells takes several days. This long induction time, and a difference in activity between GM‐CSF‐conditioned medium and GM‐CSF, together suggest that GM‐CSF stimulates WEHI 265.1 cells to secrete a factor that modulates Ii expression. These results may imply a class II‐independent function of Ii, which we discuss in this paper.
Altered interleukin‐2 receptor α‐chain is expressed in human T‐cell leukaemia virus type‐I‐infected T‐cell lines and human peripheral blood mononuclear cells of adult T‐cell leukaemia patients through an alternative splicing mechanism
Authors: HORIUCHI S. , KOYANAGI Y. , TANAKA Y. , WAKI M. , MATSUMOTO A. , ZHOU Y. W. , YAMAMOTO M. , YAMAMOTO N.
Published: November 2003

A polymerase chain reaction (PCR) method was used to detect the interleukin‐2 receptor α‐chain (IL‐2Rα) chain which lacks the conventional transmembrane (TM) domain in mRNA from human T‐cell leukaemia virus type‐I (HTLV‐I) ‐infected cell lines or peripheral blood mononuclear cells (PBMC) isolated from adult T‐cell leukaemia (ATL) patients. Primer pairs encompassing the TM domain were selected to generate a 357‐base pair (bp) fragment. A 146‐bp PCR product was observed consistently in addition to the target 357‐bp PCR product in mRNA from HTLV‐I‐infected cell lines, such as MT‐1, MT‐2, MT‐4 and in PBMC isolated from ATL patients. However, this 146‐bp PCR product was undetectable in HTLV‐I‐negative cell lines. The product was also detected in PBMC from normal individuals if activated in vitro with phytohaemagglutinin but not without stimulation. DNA sequence analyses revealed that exons from 5 to 7, which define a 211‐bp region containing the conventional TM domain, were deleted in the 146‐bp PCR product. The C‐terminal amino acid sequence starting from Gly174 of the 211‐bp‐deleted molecule was distinct from that of conventional IL‐2Rα as a result of an altered reading frame. We identified a 45 000 MW peptide generated from IL‐2Rα mRNA through this exon skip in cell lysate of MT‐1 and MT‐2 by Western blot analyses using an antibody raised against the peptides specific to an altered IL‐2Rα. Our results indicate that an altered IL‐2Rα chain is expressed in HTLV‐I‐infected T lymphocytic cell lines and in ATL patients.
CR3‐dependent phagocytosis by murine macrophages: different cytokines regulate ingestion of a defined CR3 ligand and complement‐opsonized Cryptococcus neoformans
Authors: CROSS C. E. , COLLINS H. L. , BANCROFT G. J.
Published: November 2003

Phagocytosis is a fundamental process in innate resistance to infection. We have used the pathogenic yeast Cryptococcus neoformans to study the interaction of this encapsulated organism with murine macrophages in vitro. In the absence of exogenous opsonins the encapsulated yeast is almost totally resistant to ingestion by murine macrophages. Owing to its ability to activate the alternative complement pathway, the anti‐phagocytic properties of the polysaccharide capsule can be partially overcome following opsonization in vitro with non‐immune mouse serum and subsequent phagocytosis via complement receptors. Here, we demonstrate the importance of the complement receptor type 3 (CR3) in in vitro phagocytosis of the yeast and in in vivo resistance to infection. In vitro, 70% of a population of resident murine macrophages are able to ingest C. neoformans and then only inefficiently (1–2 organisms per cell). Previously we have shown that tumour necrosis factor‐α (TNF‐α) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) efficiently enhance ingestion of serum‐opsonized encapsulated C. neoformans, and we now show that the cytokines convert a population of resident macrophages to a state where all the cells are competent for ingestion of large numbers of yeasts (6–8 per cell). We also show that these cytokines have a direct effect on CR3, as enhanced levels of complement‐opsonized sheep red blood cells (EIgMC) bind to macrophages activated in this way. However, cytokines that have previously been shown to enhance phagocytosis of EIgMC have no effect on ingestion of encapsulated C. neoformans. These results demonstrate that the cytokines regulating CR3‐dependent ingestion of C. neoformans are different to those regulating ingestion of EIgMC and reinforce the importance of studying pathogens rather than inert ligands in understanding the regulation of phagocytosis.
Autologous killing by a population of intermediate T‐cell receptor cells and its NK1.1+ and NK1.1− subsets, using Fas ligand/Fas molecules
Authors: MORODA T. , IIAI T. , SUZUKI S. , TSUKAHARA A. , TADA T. , NOSE M. , HATAKEYAMA K. , SEKI S. , TAKEDA K. , WATANABE H. , ABO T.
Published: November 2003

Self‐reactive clones, estimated by anti‐Vβ monoclonal antibodies (mAb) in conjunction with the Mls system, are confined to a population of intermediate (int) T‐cell receptor (TCR) (or CD3) cells (i.e. TCRint cells), but are not found among TCRhigh cells. The next questions to be answered are whether autologous killing is confined to TCRint cells and how such killing is mediated. In this study, 51Cr‐labelled thymocytes of syngeneic or allogeneic origin were used as target cells (4‐hr assay). When liver and splenic mononuclear cells (MNC) obtained from B6 mice were used as effector cells, prominent autologous killing was seen in liver MNC, but not splenic MNC. Such killing was not seen when thymocytes from B6‐lpr/lpr mice (i.e. Fas−) were used as target cells, nor when liver MNC from MRL‐gld/gld mice (i.e. Fas ligand−) were used as effector cells (target thymocytes of MRL‐+/+ mice). Cell separation experiments using a cell sorter revealed that autologous killing was mediated for the most part by CD3int cells, while allogeneic killing was mediated entirely by natural killer (NK) cells, TCRint cells and TCRhigh cells. Among CD3int cells, the NK1.1+ subset (i.e. NK1.1+ T cells) manifested a higher level of autologous killing than did the NK1.1− subset. Consistent with the results of a functional assay, it was found by reverse‐transcription–polymerase chain reaction (RT‐PCR) assay that CD3int cells among liver MNC showed the expression of Fas ligand mRNA, while thymocytes expressed Fas mRNA. When class I major histocompatibility complex (MHC)− thymocytes (from β2‐microglobulin‐deficient mice) were used as target cells, NK cells, but not CD3int cells, showed potent cytotoxicity. These results suggest that autologous killing is a major function of TCRint cells with self‐reactivity, and that such killing is mediated by means of Fas ligand/Fas molecules.
Differential expression of CD32 isoforms following alloactivation of human T cells
Authors: SANDILANDS G. P. , MACPHERSON S. A. , BURNETT E. R. , RUSSELL A. J. , DOWNIE I. , MACSWEEN R. N. M.
Published: November 2003

Receptors for the Fc region of immunoglobulin G (IgG) (FcγRs) exist in three main forms: membrane bound, soluble and cytoplasmic. The function of cytoplasmic FcγRs is poorly understood. We have previously demonstrated cytoplasmic FcγRII (cCD32) within most normal human peripheral blood lymphocytes (PBL), including T cells. In this study we have investigated the hypothesis that following lymphocyte activation, up‐regulation of cCD32 occurs, resulting in increased expression at the cell surface. Normal PBL were activated in vitro using a two‐way mixed lymphocyte reaction (MLR) and expression of CD32 monitored by flow cytometry and by immunoperoxidase staining using specific monoclonal antibodies and aggregated mouse IgG sub‐classes. Furthermore, we designed oligonucleotide probes specific for the three main isoforms of CD32 and looked for changes in mRNA expression throughout the MLR using an in situ hybridization technique. Increased surface expression of CD32 was found on both activated human T and B lymphocytes, but this was found only in the early stages of the MLR, on days 3 and 4, and was virtually absent by day 7. An inverse relationship between cell surface expression of CD32 and mRNA for the IIb isoforms was noted with strong mRNA expression for IIb isoforms occurring in the later stages of the MLR (days 6–7) when interleukin‐2R (IL‐2R)‐positive T cells were predominant. A soluble IgG binding factor (soluble CD32?) was also detected in the MLR culture supernatant. These observations provide support for the hypothesis that synthesis of IIb isoforms of CD32 occurs following alloantigen activation of human T lymphocytes.
Correlation between natural killer cell activation in the bone marrow and haemopoietic dysfunction following cytomegalovirus infection of mice
Authors: GIBBONS A. E. , SHELLAM G. R. , PRICE P.
Published: November 2003

We describe here the activation of natural killer (NK) cells in the bone marrows and spleens of mice infected with murine cytomegalovirus (MCMV). NK activity at these sites peaked at day 2 to 3 post‐infection (p.i.) and declined between days 6 and 10 p.i. in BALB/c and C57BL/6 mice. In BALB/c mice, the increases in NK activity coincided with depletion of colony‐forming units of the granulocyte–monocyte lineage (CFU‐GM) from the marrow. CFU‐GM depletion in MCMV‐infected C57BL/6 mice was less severe, despite the presence of activated NK cells in the marrow. Treatment of BALB/c mice with anti‐asialo GM1 prior to MCMV infection resulted in less severe CFU‐GM depletion at day 2 p.i. than infection with MCMV alone. When homozygous C57BL/6 or CBA/CaH bg/bg mice were infected with MCMV, depletion of marrow CFU‐GM was more severe than in their heterozygous littermates. Finally, we observed some inhibition of colony formation when marrow cells from MCMV‐infected and uninfected BALB/c donors were mixed and incubated prior to the CFU‐GM assay. These results suggest that activated NK cells may contribute to depletion of haemopoietic cells soon after MCMV infection of BALB/c mice, but may limit the loss of these cells in C57BL/6 and CBA/CaH mice.
Inactivation and proteolytic degradation of perforin within lytic granules upon neutralization of acidic pH
Authors: KATAOKA T. , TOGASHI K. , TAKAYAMA H. , TAKAKU K. , NAGAI K.
Published: November 2003

In our recent studies, an inhibitor of vacuolar‐type H+‐ATPase, concanamycin A (CMA) has been shown to neutralize acidic pH in vacuolar organelles, including lytic granules, and to decrease the perforin content markedly. In the present paper, we have further investigated the role of acidification in perforin storage by using CMA. In CD8+ cytotoxic T‐lymphocyte (CTL) clones, the amount of perforin decreased rapidly at 30–90 min but no more decrease occurred at 90–120 min after the addition of CMA. Since exposure to actinomycin D, cycloheximide, or brefeldin A failed to reduce the perforin content, the perforin decrease in CMA‐treated cells seems to be largely due to a reduction in the perforin already stored in lytic granules, rather than to the inhibition of the de novo synthesis or the intracellular glycoprotein transport of perforin. Diisopropylfluorophosphoridate (DFP) markedly antagonized the decrease in the perforin content in CMA‐treated cells, while other protease inhibitors, i.e. antipain, E‐64, leupeptin, pepstatin A and phenylmethylsulphonyl fluoride, did not. Nevertheless, DFP hardly reversed the abrogation of the killing activity by CMA. Indeed, the lytic granules prepared from DFP plus CMA‐treated cells showed only a marginal level of haemolytic activity. In cell‐free experiments using perforin‐enriched granule fractions, acidic pH completely blocked the perforin activity. Under the acidic conditions, perforin was more resistant to an inactivation by calcium when exposed to calcium prior to the haemolysis test. Thus, these data suggest that perforin is primarily inactivated, possibly in a calcium‐dependent manner, and is subsequently hydrolysed by DFP‐sensitive proteases in the lytic granules at neutral pH. We conclude that acidic pH plays an essential role to maintain the integrity of perforin within the lytic granules.
Identical expression of CD45R isoforms by CD45RC+‘revertant’ memory and CD45RC+ naive CD4 T cells
Authors: HARGREAVES M. , BELL E. B.
Published: November 2003

Naive and memory CD4 T cells are frequently defined by exon‐specific monoclonal antibodies (mAb) which stain (or not) high‐ or low‐molecular‐weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a ‘memory’ phenotype (CD45RA−, RB−, RC−, or CD45RO+) may revert (‘revertants’) and re‐express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T‐cell development. Furthermore, the picture may be incomplete since an exon‐specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti‐CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3‐exon isoform ABC, the 2‐exon isoforms AB and BC, and the null isoform O. Both immature CD45RC− (i.e. CD4+8−‘single positive’ thymocytes, and peripheral Thy‐1+ recent thymic emigrants) and mature CD45RC−‘antigen‐experienced’ CD4 T cells had message for single‐exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen (‘truly naive’) and CD45RC+ antigen‐experienced revertant memory T cells. This observation has implications for understanding long‐term immunological memory.
Immune response to murine cell lines of glial origin transplanted into the central nervous system of adult mice
Authors: TERRY L. A. , USHERWOOD E. J. , LEES S. , MACINTYRE N. , NASH A. A.
Published: November 2003

Temperature‐sensitive simian virus 40 (SV40) T antigen‐transformed central nervous system (CNS)‐derived murine cell lines were used to analyse the host response to transplantation in the mouse adult brain. The cell lines were shown to be susceptible to immune recognition in vitro by cytotoxic effector cells indicating that tissue‐specific privilege was not in operation. Histological examination at time points post‐implantation showed characteristic responses similar to those seen during graft rejection. Astrocytosis and up‐regulation of major histocompatibility complex (MHC) class I and MHC class II activation of resident microglia and recruitment of macrophages were observed in both allogeneic and syngeneic hosts 10 days post‐implantation suggesting a trauma‐induced response. However, the response in allogeneic hosts was more widespread and evident when the syngeneic responses had returned to normal levels. Evidence of T‐cell infiltration was also more pronounced in the allogeneic hosts. Despite quite extensive host reactions to these cellular grafts at early time‐points the implants appeared to survive in the host CNS long after the responses had abated and could be detected at the maximum time‐point studied of 40 days.
T‐cell epitopes recognized within the 65 000 MW hsp in patients‘qc with IgA nephropathy
Authors: WARR K. , FORTUNE F. , NAMIE‡ S. , WILSON A. , SHINNICK§ T. , VAN DER ZEE¶ R. , WILLIAMS G. , LEHNER T.
Published: November 2003

IgA nephropathy (IgAN) is the commonest cause of glomerulonephritis and clinical exacerbation of IgAN is frequently associated with mucosal infection. T‐cell receptorγδ (TCRγδ+) cells are increased in both the circulation and in renal biopsies of patients with progressive IgAN. We examined the hypothesis that specific peptides within the 65 000 MW heat‐shock protein (hsp) might stimulate TCRγδ cells and play a part in the immunopathogenesis of IgAN. We studied T‐cell proliferative responses stimulated by overlapping peptides derived from the sequence of mycobacterial 65 000 MW hsp. Three T‐cell epitopes have been identified (peptides 51–65, 71–85 and 281–295). The three peptides have a synergistic effect and they stimulate significantly higher proliferation of T cells in patients with IgAN than in disease or healthy controls. This response was inhibited by monoclonal antibodies (mAb) to TCRγδ+ and human leucocyte antigen (HLA) class I, but not by mAb to HLA class II. The involvement of TCRγδ+ cells was confirmed by up‐regulation of the proportion of TCRγδ+ cells when stimulated with the three specific peptides. We suggest that IgAN might be associated with mucosal infection by a variety of micro‐organisms and that peptides within the microbial hsp cross‐react with the homologous human hsp which may stimulate TCRγδ+ cells and play a part in the pathogenesis of IgAN.
Histamine involvement in UVB‐ and cis‐urocanic acid‐induced systemic suppression of contact hypersensitivity responses
Authors: HART P. H. , JAKSIC A. , SWIFT G. , NORVAL M. , EL‐GHORR A. A. , FINLAY‐JONES J. J.
Published: November 2003

Studies in experimental models have implicated histamine and prostanoids in ultra‐violet B (UVB)‐ and cis‐urocanic acid (UCA)‐induced systemic immunosuppression. This study examined he hypothesis that UVB irradiation and cis‐UCA suppressed contact hypersensitivity responses to hapten by induction of histamine, which in turn evoked a prostanoid‐dependent component of immunosuppression. BALB/c mice were administered with a cis‐UCA monoclonal antibody, a combination of histamine types 1 and 2 receptor antagonists, or indomethacin. Mice were sensitized to 2,4,6‐trinitrochlorobenzene (TNCB) on their ventral surface 5 days after UVB irradiation, or cis‐UCA or histamine administration. Ears were challenged with TNCB 5 days later. Cis‐UCA antibody inhibited the suppressive effects of UVB by approximately 60% and confirmed that suppression of contact hypersensitivity responses by UVB was due, at least in part, to mechanisms involving cis‐UCA. Histamine suppressed contact hypersensitivity responses and the effects of cis‐UCA and histamine were not cumulative, suggesting that cis‐UCA and histamine signal largely through the same pathway. The immunosuppressive effects of histamine were not affected by the cis‐UCA antibody, consistent with the model that histamine acts downstream of cis‐UCA. Administration of histamine receptor antagonists and indomethacin each approximately halved the UVB‐ and cis‐UCA‐induced systemic suppression of contact hypersensitivity responses. The effects of the reagents that inhibited the action of histamine and prevented prostanoid production were not cumulative, and suggested involvement in the same pathway. These results support the involvement of cis‐UCA, histamine and prostanoids, in a sequence, in UVB‐induced systemic suppression of contact hypersensitivity responses.
The effects of dexamethasone and chlorpromazine on tumour necrosis factor‐α, interleukin‐1β, interleukin‐1 receptor antagonist and interleukin‐10 in human volunteers
Authors: BLEEKER M. W. P. , NETEA M. G. , KULLBERG B. J. , VAN DER VEN‐JONGEKRIJG J. , VAN DER MEER J. W. M.
Published: November 2003

Tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) are pro‐inflammatory cytokines that play an important role in severe infections, whereas IL‐1 receptor antagonist (IL‐1ra) and IL‐10 are anti‐inflammatory cytokines that counteract their effects. Chlorpromazine and dexamethasone protect mice against lethal endotoxaemia by decreasing circulating concentrations of TNF‐α and IL‐1β. We investigated whether administration of chlorpromazine or dexamethasone to human volunteers is able to modulate the lipopolysaccharide (LPS)‐stimulated cytokine production capacity in whole blood. Blood samples were taken before and several time‐points after medication. Circulating cytokine concentrations were low in all samples. LPS‐induced TNF‐α and IL‐1β production in whole blood was inhibited by dexamethasone treatment, while chlorpromazine had no effect. When peripheral blood mononuclear cells were stimulated in vitro with LPS, the addition of chlorpromazine (1–100 ng/ml) had no modulatory action on TNF‐α, IL‐1β, IL‐1ra or IL‐10 synthesis. The chlorpromazine concentrations measured in circulation of volunteers were eight to 40 times lower than the concentrations shown to be effective in mice. In conclusion, chlorpromazine inhibits TNF‐α and IL‐1β production in mice at concentrations that cannot be reached in humans, thus precluding its usage in clinical anti‐cytokine strategies. In contrast, dexamethasone is an effective inhibitor of pro‐inflammatory cytokine production.
Human monocyte‐derived macrophages and dendritic cells are comparably effective in vitro in presenting HLA class I‐restricted exogenous peptides
Authors: TOUJAS L. , DELCROS J.‐G. , DIEZ E. , GERVOIS N. , SEMANA G. , CORRADIN G. , JOTEREAU F.
Published: November 2003

Recent experimental data have shown that mice could be immunized efficiently, in particular against cancer, by the injection of antigen‐loaded dendritic cells (DC) or macrophages (MPH). In the present work, these two antigen‐presenting cells (APC) were prepared in humans from circulating mononuclear cells (MNC). MPH were obtained from MNC that were cultured in hydrophobic plastic bags and purified by elutriation. DC were from the culture of adherent elutriation‐purified monocytes in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4). The two APC were prepared in parallel from the same donors and their phenotype and antigen‐presenting capacity were compared. DC differed from MPH by a higher expression of HLA‐DR and CD23 and a lower expression of CD14, CD64 and of adhesion molecules. DC and MPH were comparably effective in (a) enhancing the mitotic response of autologous lymphocytes to immobilized anti‐CD3 (accessory function); (b) presenting melanoma peptides to specific cytotoxic T lymphocyte (CTL) clones; and (c) stimulating the generation of CTL directed against a myxovirus influenza peptide. However, DC were more effective than MPH in inducing the mitotic response of allogeneic peripheral blood leucocytes (PBL), possibly because of their higher expression of HLA class II molecules. In conclusion, DC and MPH prepared from blood MNC did not differ substantially in their ability to activate HLA class I‐restricted T‐cell responses by exogenous peptide presentation.