bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a novel antigenic structure of the human receptor for interleukin‐6 involved in the interaction with the glycoprotein 130 chain
Authors: GAILLARD J. P. , LIAUTARD J. , MANI J. C. , FERNANDEZ SUAREZ J. M. , KLEIN B. , BROCHIER J.
Published: November 2003

The receptor for interleukin‐6 (IL‐6) is characterized by a ligand‐binding glycoprotein 80 (gp80) transmembrane chain (IL‐6R) which associates with a signal‐transducer gp130 chain. We previously raised a series of monoclonal antibodies (mAb) recognizing different epitopes of the human IL‐6R and interfering with the function of the receptor. One of them, M182, was able to diminish the proliferation of IL‐6‐dependent plasmacytoma cell lines although it was found unable to inhibit the binding of IL‐6 to its receptor. Using an enzyme‐linked immunosorbent assay for measuring the binding of IL‐6–IL‐6R to the gp130 chain, we showed that M182 was directed against a structure directly involved in the IL‐6R/gp130 interaction. M182 was able to potentiate the inhibitor effect of anti‐IL‐6R mAB which interfere with the binding of IL‐6, leading to complete inhibition of the proliferation of IL‐6‐dependent cell lines. M182 was also found to synergize with inhibitory anti‐IL‐6 mAb. Therefore this structure appears to be an important regulatory domain of the IL‐6R and a valuable target for inhibiting IL‐6 signalling.
Priming differentially regulates neutrophil adhesion molecule expression/function
Authors: CONDLIFFE A. M. , CHILVERS E. R. , HASLETT C. , DRANSFIELD I.
Published: November 2003

Lung injury in a variety of disease states is critically dependent on neutrophil‐mediated inflammatory responses. Neutrophil recruitment to sites of infection or tissue damage requires co‐ordinated regulation of neutrophil adhesion and activation status. We have examined the effects of treatment of human peripheral blood neutrophils with priming agents [lipopolysaccharide (LPS), tumour necrosis factor‐α (TNF‐α) and platelet‐activating factor (PAF)] upon expression of CD11a, CD11b, CD11c, CD35 and CD62‐L and CD11b function to assess whether subtle regulation of neutrophil adhesion potential accompanies augmented formyl‐methionyl‐leucyl‐phenylalanine‐stimulated superoxide production. We have found that there are differential effects of ‘priming’ concentrations of these agents. For LPS, CD62L loss occurs in the absence of changes in CD11b, whereas for PAF, CD11b up‐regulation occurs in the absence of detectable loss of CD62‐L. However, for TNF‐α, decreased expression of CD62‐L occurs concomitantly with increased expression of CD11b. In addition, we have shown that priming agents augment CD11b functional activity in a manner that parallels the priming of the respiratory burst. Thus, priming agents may differentially regulate neutrophil adhesive capacity and data presented in this manuscript suggest that the increased effector cell function observed in primed cells may be associated with a distinct repertoire of potential adhesive interactions.
Two mechanisms for platelet‐mediated killing of tumour cells: one cyclo‐oxygenase dependent and the other nitric oxide dependent
Authors: OKADA M. , SAGAWA T. , TOMINAGA A. , KODAMA T. , HITSUMOTO Y.
Published: November 2003

We have tried to identify the cytotoxic effectors in platelet‐mediated tumour cell killing, using two tumour cell lines K562 (a chronic myelogenic leukaemic cell line) and LU99A (a lung cancer cell line), which are both sensitive to platelet cytotoxicity. Cyclo‐oxygenase inhibitors, acetylsalicylic acid (ASA) and indomethacin, effectively inhibited the platelet‐mediated killing of K562 cells, but not that of LU99A cells. In contrast, inhibitors of the nitric oxide (NO) pathway, NG‐nitro‐L‐arginine (L‐NA), haemoglobin and methylene blue, reduced the cytotoxic activity of platelets against LU99A, but not against K562. Synthetic analogues of platelet–cyclo‐oxygenase products thromboxane A2/prostaglandin H2 (TXA2/PGH2) exerted cytotoxicity against K562 cells but not against LU99A cells. Electron microscopic study showed that TXA2/PGH2 analogues induced bleb formation and disruption of the plasma membrane of K562 cells. K562 cells enhanced the production of TXA2 by platelets, as inferred from the accumulation of thromboxane B2 (TXB2), a spontaneous hydrolysis product of TXA2. LU99A cells had no such effects. These results indicate that platelets kill these two tumour cell lines through different mechanisms. In K562, the cyclo‐oxygenase products TXA2/PGH2 possibly play a significant role, but in LU99A the NO pathway seems to be involved.
Diabetes‐prone NOD mice are resistant to Mycobacterium avium and the infection prevents autoimmune disease
Authors: BRÁS A. , ÁGUAS A. P.
Published: November 2003

It was recently proposed that the diabetes genes of non‐obese diabetic (NOD) mice are linked to the Bcg gene that is associated with resistance to infection by mycobacteria; however, it has not been established whether NOD mice are resistant or susceptible to the infection, although there are previous investigations on response of NOD mice to other intracellular parasites (e.g. Kaye et al., Eur. J. Immunol.22: 357–364). We have investigated here this question, as well as the consequences of mycobacterial infection on the natural history of murine diabetes. Female NOD mice were intraperitoneally infected with 108 viable bacilli of Mycobacterium avium at 2 months of age, i.e. before the mice show diabetes; they were studied up to the sixth month of age (when more than half of untreated female NOD mice show glycosuria). To determine whether NOD mice were susceptible or resistant to M. avium infection, we have compared the kinetics of bacterial growths in liver and spleen of the mice with those determined in M. avium‐susceptible (BALB/c) and resistant (C3H) strains of mice. NOD mice were able to control the M. avium infection, following a pattern similar to that observed in infected C3H mice. The mycobacterial infection prevented the expression of diabetes in all of the infected NOD mice and it also decreased the incidence of proteinuria in the treated mice. The infected NOD mice showed a marked enhancement in antibodies against the 65 000 mycobacterial antigen (heat‐shock protein (hsp) 65) up to the second month of infection and these elevated titres slowly decreased in the following months; anti‐hsp 65 antibodies were not detected in age‐matched controls. This is the first demonstration that NOD mice are naturally resistant to mycobacterial infection, and we reinforce evidence on the role of immune response triggered by mycobacteria and its hsp 65 antigen in prevention of diabetes in NOD mice.
The role of CD4 molecules in the induction phase of contact hypersensitivity cytokine profiles in the skin and lymph nodes
Authors: FUJISAWA H. , KONDO S. , WANG B. , SHIVJI G. M. , SAUDER D. N.
Published: November 2003

We have previously demonstrated that CD4 gene‐targeted mutant mice (CD4− mice) demonstrate hyporesposiveness in contact hypersensitivity (CHS) suggesting that CD4 molecules are required for optimal induction of CHS. In the present study, we wished to examine the mechanisms of this hyporesponsiveness, in particular, we examined whether cytokines were altered in the skin and lymph nodes of CD4− mice following exposure to the contact allergen dinitrofluorobenzene (DNFB). Cytokine mRNA in the ear skin and draining lymph nodes (DLN) were examined by reverse transcription–polymerase chain reacion (RT–PCR) at various times after sensitization. Skin cytokine patterns revealed that in normal mice, interleukin (IL)‐2, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α mRNA levels increased at 12 hr sensitization, whereas these cytokines were below the level of detection in CD4− mice. In the DLN of normal mice following the hapten application, sequential upregulation of cytokine mRNA including IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐10, IFN‐γ and TNF‐α was found. No change was seen for IL‐1α, IL‐1β, IL‐10 and TNF‐α and IL‐2, IL‐4 and IFN‐γ mRNA levels were below the level of detection in DLN from CD4– mice following the hapten application. However, IL‐1β, IL‐2 and TNF‐α mRNA levels of lymph node cells from CD4− mice could be upregulated by phorbol myristate acetate in vitro. Flow cytometry study has revealed that the number of Langerhans’ cells (LC) in DLN of CD4− mice was similar to that of normal mice, thus, inferring that the alterations of cytokine milieu in the ear skin did not have a significant effect on LC migration to DLN. These results suggest that CD4 molecules are crucial for the induction of certain cytokines in the skin and in inducing sequential cytokine signals in DLN required for optimal development of CHS, but that these changes in cytokines do not effect LC migration.
Distribution of β7 integrins in human intestinal mucosa and organized gut‐associated lymphoid tissue
Authors: FARSTAD I. N. , HALSTENSEN T. S. , LIEN B. , KILSHAW P. J. , LAZAROVITZ A. I. , BRANDTZAEG P.
Published: November 2003

Two alternative integrins involved in mucosal homing (α4β7) or epithelial retention (αEβ7) of lymphocytes were examined in the human gut. The distribution of the β7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by αEβ7+ cells but weakly by α4β7+ cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T‐cell receptor (TCR)γ/δ+, and CD3− cells, expressed αEβ7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B‐lineage cells (CD19+) and macrophages (CD68+) were negative. In gut‐associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (<5%) cells were positive for αEβ7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30–50%) expressed α4β7 (mAb Act‐1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of α4β7 as in the lamina propria whereas relatively few B lymphocytes (<50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for α4 and the former three subsets as well as appendix CD8+ cells also for β7 while only lamina propria CD8+ cells had mRNA for αE. Together, the results suggested that αEβ7 and α4β7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly α4β7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because αEβ7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of β7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle‐associated epithelium of GALT expressed αEβ7, perhaps reflecting epithelial interaction during primary immune responses.
Development and cytolytic function of intestinal intraepithelial T lymphocytes in antigen‐minimized mice
Authors: KAWAGUCHI‐MIYASHITA M. , SHIMIZU K. , NANNO M. , SHIMADA S. , WATANABE T. , KOGA Y. , MATSUOKA Y. , ISHIKAWA H. , HASHIMOTO K. , OHWAKI M.
Published: November 2003

Intraepithelial T lymphocytes in the small intestine (IEL) consist of αβ T‐cell receptor (TCR)‐bearing T cells (αβ‐IEL) and γδ TCR‐bearing T cells (γδ‐IEL). Development and cytolytic activation of αβ‐IEL sharply attenuate in germ‐free (GF) mice fed a natural diet (Nat‐GF), but the number and cytotoxicity of γδ‐IEL are comparable between conventional (CV) and Nat‐GF mice. In this report, we compared the properties of IEL in Nat‐GF mice and GF mice fed antigen‐minimized diet (AgM‐GF mice) of C57BL/6 strain to evaluate an influence of gut antigenic load on IEL development. Numbers of αβ‐IEL and γδ‐IEL in AgM‐GF mice were less by 1.9‐ and 1.4‐fold than those in Nat‐GF mice, respectively. Significant decreases in the proportions of CD4+8−, CD4−8αβ+, and CD4+8+ subsets and a resultant increase in the ratio of CD4−8αα+ subset were evident in αβ‐IEL of Nat‐GF mice compared with CV mice, but the subset constitution of αβ‐IEL was similar between Nat‐GF and AgM‐GF mice. In contrast, relative composition of γδ‐IEL was not different between CV, Nat‐GF, and AgM‐GF mice. αβ‐IEL displayed low cytolytic activity in Nat‐GF mice and were almost deprived of their cytotoxicity under the antigen‐minimized condition. While γδ‐IEL were strongly cytolytic in Nat‐GF mice their cytolytic activity was remarkably reduced in AgM‐GF mice. These results indicate that γδ‐IEL are activated independently of microbial colonization in the gastrointestinal tract but their activation occurs in response to the exogenous antigenic substances other than live micro‐organisms.
CD7‐negative T cells represent a separate differentiation pathway in a subset of post‐thymic helper T cells
Authors: REINHOLD U. , LIU L. , SESTERHENN J. , ABKEN H.
Published: November 2003

The absence of CD7 protein and the corresponding mRNA is a stable feature in a subset of normal circulating CD4+ memory T cells. It is still unresolved whether the CD7− subset represents a specific T‐cell lineage. Here we show that repeated stimulation of highly purified CD4+ CD45RA+ CD45RO− naive T cells in vitro leads to the development of a distinct memory subset that is defined by the expression versus non‐expression of the CD7 antigen. Comparing different T‐cell activation pathways (TCR/CD3, CD2), we observed that alternative signals were critically involved in the development of CD4+ CD7− T cells. Peak mean numbers of CD7− memory cells occurred after 3–5 cycles of restimulation in vitro. Naive T cells that had undergone repeated stimulations were harvested and sorted into CD7+ and CD7− subsets. The vast majority (> 97%) of CD7+ T cells retained their expression, whereas the CD7− population did not re‐express the antigen during further propagation of separated T‐cell subsets. In CD7− cells no CD7 mRNA was monitored, indicating transcriptional regulation of CD7 expression. Certain differentiation‐related antigens, including the cutaneous lymphocyte antigen CLA, were preferentially expressed on CD7− T cells. We suggest that absence of CD7 expression in a subset of CD4+ memory cells reflects a separate and stable differentiation state occurring late in the immune response. These T cells may represent the physiological counterpart of malignant T cells in certain forms of cutaneous T‐cell lymphoma.
Down‐regulation of cyclooxygenase‐2 (COX‐2) by interleukin‐1 receptor antagonist in human monocytes
Authors: PORRECA E. , REALE M. , DI FEBBO C. , DI GIOACCHINO M. , BARBACANE R. C. , CASTELLANI M. L. , BACCANTE G. , CONTI P. , CUCCURULLO F.
Published: November 2003

Cyclooxygenase (COX) is the key rate‐limiting enzyme in the synthesis of prostanoids from arachidonic acid. Two isoforms of COX have been described in mammalian cells, referred to as cyclooxygenase‐1 (COX‐1) and cyclooxygenase‐2 (COX‐2). COX‐1 is a constitutively expressed enzyme; COX‐2 is an inducible enzyme that appears to be expressed in inflamed tissue and following exposure to growth factors or cytokines, such as interleukin‐1 (IL‐1). The aim of the present study was to test if the antagonism on the binding of IL‐1 to its cell‐surface receptor by human recombinant IL‐1 receptor antagonist (hrIL‐1ra) may control the COX mRNA expression and prostaglandin E2 (PGE2) production by human monocyte cultures. Northern blot studies showed that hrIL‐ra (500 ng/ml) had a strong inhibitory effect on inducible COX activity. The effect was evident after 6 hr incubation (2.7‐fold decrease of mRNA COX‐2 transcripts); and about a threefold decrease at 24 hr incubation. A non‐significant effect was observed with COX‐1 transcripts. Induced PGE2 production by monocyte cultures treated with lipopolysaccharide (LPS) or interleukin‐1β (IL‐1β) was strongly inhibited in the presence of hrIL‐1ra (500 ng/ml). In addition, a significant inhibition of COX‐2 protein expression, as evaluated by Western blotting, was also observed. These data suggest that hrIL‐1ra may be the key mediator in the down‐regulation of the COX‐2 inducible pathway.
Fibroblasts prevent apoptosis of IL‐2‐deprived T cells without inducing proliferation: a selective effect on Bcl‐xL expression
Authors: GOMBERT W. , BORTHWICK N. J. , WALLACE D. L. , HYDE H. , BOFILL M. , PILLING D. , BEVERLEY P. C. L. , JANOSSY G. , SALMON M. , AKBAR A. N.
Published: November 2003

The apoptosis of human cytokine‐deprived activated T cells can be prevented by a soluble mediator secreted by fibroblasts, epithelial and endothelial cells, and this rescue occurs with fibroblasts from different species. Fractionation of WI38 fibroblast‐conditioned medium indicated that the survival‐promoting agent(s) were >30 000 MW. The continuous presence of the survival factor was required for prevention of apoptosis, which did not involve the induction of proliferation. Nevertheless, the co‐cultured T cells remained in a primed state. The expression of the apoptosis‐inducing proteins Bax and CD95 (Fas/Apo‐1) was either unchanged or slightly increased in fibroblast‐rescued T cells, suggesting that constraints on survival still existed after co‐culture. A fundamental observation in the present study was that although Bcl‐2 was reduced, the levels of Bcl‐xL was maintained in cytokine‐deprived T cells by fibroblast co‐culture. This suggests that fibroblasts and/or other stromal cells may promote activated T‐cell survival by a selective effect on Bcl‐xL expression, which is consistent with histological examination of activated T cells within lymphoid tissue in vivo. The rescued T cells could be re‐activated by CD3 antibody, but only in the presence of CD28 co‐stimulation, which induced both Bcl‐2 and Bcl‐xL expression and also proliferation. Thus, survival signals from stromal cells in tissue microenvironments may enable activated T‐cell persistence in a primed but quiescent state, and our data suggest that the regulation of Bcl‐xL expression may be central in this process. The further characterization of this process is essential to clarify how signals from stromal cells can influence the resolution and/or chronicity of immune responses in different tissues in vivo.
Peptides isolated from random peptide libraries on phage elicit a neutralizing anti‐HIV‐1 response: analysis of immunological mimicry
Authors: LUNDIN K. , SAMUELSSON A. , JANSSON M. , HINKULA J. , WAHREN B. , WIGZELL H. , PERSSON M. A. A.
Published: November 2003

Peptides binding to a murine, human immunodeficiency virus type 1 (HIV‐1) neutralizing monoclonal antibody (F58/H3) were isolated from two random peptide libraries expressed on the surface of phage. The antibody was originally elicited by immunization with HIV‐1 envelope protein gp120LAI, and has previously been shown to interact with the ‐I–GPGRA‐ motif of the V3 loop. The peptide libraries consisted of nine or 15 random amino acid residues flanked by two cysteines, and fused to the amino terminal end of the cpIII protein on the filamentous phage. Selection of specific peptides was carried out in three rounds, with decreasing antibody concentration. An expected peptide motif ‐GPGRA‐, a similar segment, ‐GPAR‐, and two unrelated motifs ‐FRLLG‐ and ‐WRM/ALG‐ were selected. Binding of antibody was tested both to synthetic peptides in solution, and the corresponding peptide on phage. The GPXR motifs bound in both formats, while the FRLLG bound antibody only when present on the phage. The reactivity of peptides on phage was highly dependent on an intact disulphide bond between the cysteines flanking the peptide. The molecular mimicry of the found motifs was tested by immunizing mice and rabbits with conjugated synthetic peptides or peptide on phage. In mice, peptide‐specific antisera were raised, but no reactivity to the whole protein (gp120) was detected. In rabbits, however, this was accomplished with the ‐GPGRA‐ containing peptide when present on phage. In addition, this antisera precipitated virus particles, and neutralized HIV‐1SF2 virus in vitro.
Increased clearance of IgG in mice that lack β2‐microglobulin: possible protective role of FcRn
Authors: ISRAEL E. J. , WILSKER D. F. , HAYES K. C. , SCHOENFELD D. , SIMISTER N. E.
Published: November 2003

The mechanisms that regulate immunoglobulin G (IgG) catabolism are little understood. We have previously found unusually low IgG concentrations in sera of mice homozygous for a targeted disruption of the β2‐microglobulin gene. We therefore investigated whether this might result, at least in part, from increased clearance of IgG from the systemic circulation in mice lacking β2‐microglobulin. We compared the half‐lives of radiolabelled mouse IgG1 injected intravenously into β2‐microglobulin−/− mice and wild‐type or heterozygous siblings. The clearance of 125I‐labelled IgG1 was strikingly more rapid in the mice lacking β2‐microglobulin. β2‐microglobulin−/− mice lack functional molecules of the MHC class I‐related Fc receptor, FcRn. Some mutations in mouse IgG1 that increase its clearance have recently been shown to prevent binding to FcRn in the gut. To determine whether the slower degradation of immunoglobulin in mice with β2‐microglobulin correlated with the ability of the antibody to bind FcRn, we measured the clearance of chicken IgY, which does not bind this receptor. The 125I‐labelled IgY was catabolized equally rapidly in β2‐microglobulin‐deficient and wild‐type mice. We compared the half‐lives of the four subclasses of mouse IgG in β2‐microglobulin−/−, +/−, and +/+ mice to determine whether the difference we had noted for IgG1 was peculiar to this subclass. The 125I‐labelled IgG of all subclasses, with the possible exception of IgG2b, was degraded more rapidly in the β2‐microglobulin‐deficient mice than in heterozygous or wild‐type siblings. These data suggest that FcRn can protect IgG from degradation, and is therefore important in maintaining IgG levels in the circulation.
Inflammation‐induced changes in the phenotype and cytokine profile of cells migrating through skin and afferent lymph
Authors: EGAN P. J. , KIMPTON W. , SEOW H.‐F. , BOWLES V. M. , BRANDON M. R. , NASH A. D.
Published: November 2003

In the present study, we have localized cytokine‐secreting cells within an ectoparasite‐induced inflammatory lesion and monitored the phenotype and cytokine profile of cells migrating from the inflammatory lesion to the local draining lymph node via the afferent lymphatics. Interleukin (IL)‐8‐producing cells were first detected in skin within 6 hr of infection, with increased numbers observed at 24 and 48 hr post infection. While these cells were concentrated within the neutrophil influx, adjacent to disrupted epidermis; they were also found scattered throughout the surrounding dermis in areas where significant cellular infiltration was not apparent. IL‐1α‐ and IL‐1β‐producing cells could not be detected until 24 hr after infection and were restricted to areas of intense neutrophil accumulation. Concurrent with the onset of inflammation was a threefold increase in the total number of cells migrating through the draining afferent lymph. This increase in cellularity was due primarily to increased migration of CD4 and γδ T cells. Cytokine mRNA synthesis by migrating afferent lymph cells was examined by reverse transcription–polymerase chain reaction (RT–PCR) analysis of RNA extracted prior to, and at regular intervals during the course of the inflammatory response. IL‐1β and IL‐8, but not IL‐1α or IL‐6 mRNA, was detected in migrating afferent lymph cells. Tumour necrosis factor (TNF)‐α‐specific mRNA was present in migrating afferent lymph cells at all time points both prior to, and following infection. Soluble IL‐8 protein, but not IL‐1α, IL‐1β or TNF‐α protein, could be detected in lymph, with the amount of IL‐8 detected increasing as the infection progressed. mRNA coding for cytokines associated with T‐cell activation, such as IL‐2, IL‐4 or interferon (IFN)‐γ, was also detected in migrating cells, although the cytokine profiles of different experimental animals were extremely variable.
A new immunosuppressant, FTY720, induces bcl‐2‐associated apoptotic cell death in human lymphocytes
Authors: SUZUKI S. , LI X.‐K. , ENOSAWA S. , SHINOMIYA T.
Published: November 2003

FTY720 is a unique immunosuppressive drug produced by modification of a metabolite from Isaria sinclairii. In vitro treatment of human mononuclear cells with FTY720 resulted in a dose‐dependent reduction of cell viability. These treated cells demonstrated characteristic DNA ladder formation on agarose gel electrophoresis. Jurkat cells transfected with human bcl‐2 gene were resistant to FTY720; their neo type was susceptible to the drug. A rapid acceleration of cell death in human mononuclear cells was seen as early as 2 hr after incubation with FTY720. The intracellular Bax protein increased remarkably 1 hr after the culture; it markedly decreased in the surviving cells at 2 and 3 hr. Coincidental to the Bax decrease, Bcl‐2 progressively decreased beginning 2 hr after the culture. Thus, the ratio of Bcl‐2 to Bax was decreased by the enhanced expression of Bax immediately after FTY720‐treatment, resulting in rapid cell death acceleration. The surviving cells (FTY720‐resistant cells) at 2 and 3 hr after culture showed a similar ratio of Bcl‐2 to Bax as was observed in the control cells. These results suggest that FTY720 displays bcl‐2‐associated apoptotic cell death in human mononuclear cells.
Microbial colonization influences composition and T‐cell receptor Vβ repertoire of intraepithelial lymphocytes in rat intestine
Authors: HELGELAND L. , VAAGE J. T. , ROLSTAD B. , MIDTVEDT T. , BRANDTZAEG P.
Published: November 2003

Studies in mice have shown that the composition of intestinal intraepithelial lymphocytes (IEL) may be markedly altered by gut microbial colonization. Such modulation was studied in a rat model by the use of germ‐free and conventionalized animals from which IEL from the small intestine were isolated and analysed by flow cytometry. Conventionalization caused expansion as well as phenotypic alterations of T‐cell receptor (TCR) α/β+ IEL in that the proportions of CD4+ and CD8αβ+TCRα/β+ cells were increased, while the double negative (CD4− CD8−) fraction was reduced. Microbial colonization also influenced the TCR Vβ repertoire of CD8+ IEL in that the proportions of Vβ8.2+ and Vβ10+ cells were increased, whereas Vβ8.5+ and Vβ16+ cells were relatively decreased. Moreover, conventionalization influenced the levels of TCR cell surface expression in the same Vβ subsets. Three‐colour flow‐cytometric analysis demonstrated that skewing of the Vβ repertoire was most pronounced in the CD8αα+ subset, although the numerical increase of IEL mainly included the CD8αβ+ subset. In contrast to IEL, the TCR Vβ repertoire in mesenteric lymph nodes was unchanged after intestinal colonization. These results confirm that TCRα/β+ IEL subpopulations respond dynamically to the microbial gut flora and suggest that their Vβ repertoire can be shaped by luminal microbial antigens.
The adjuvant effect of a non‐toxic mutant of heat‐labile enterotoxin of Escherichia coli for the induction of measles virus‐specific CTL responses after intranasal co‐immunization with a synthetic peptide
Authors: PARTIDOS C. D. , PIZZA M. , RAPPUOLI R. , STEWARD M. W.
Published: November 2003

The intranasal route has been shown to be effective for immunization. However, immunization via this route may require the use of potent and safe adjuvant. The construction of non‐toxic mutants of heat labile enterotoxin of Escherichia coli (LT), which is a potent mucosal adjuvant, is a major breakthrough for the development of mucosal vaccines. In this study we have assessed the ability of an LT mutant (LTK63) to act as an adjuvant following intranasal co‐immunization with a peptide corresponding to a measles virus cytotoxic T lymphocyte (CTL) epitope. LTK63 was more effective at potentiating the in vivo induction of peptide‐specific and measles virus‐specific CTL responses than was administration of the peptide in saline. A concentration of 10 μg/dose of LTK63 was found to be the most effective in potentiating the in vivo priming of peptide‐specific and measles virus‐specific CTL responses. These findings highlight the potential of the non‐toxic mutant of LT as a safe mucosal adjuvant for use in humans.
Mucosal immunology: from bench to the bedside and beyond
Authors: FERGUSON A.
Published: November 2003

This paper, written by the President of the Society for Mucosal Immunology, marks the 9th International Congress of Mucosal Immunology, in Sydney: Mucosal Solutions. Current molecular, cellular and animal work in mucosal immunity has great potential when applied to issues of human and animal health. However, practical and technical problems in the transfer of theoretical concepts into clinically based research must not be underestimated. Ideally, studies in disease need to be designed and run jointly by clinicians and scientists, as illustrated by examples drawn from Crohn's disease. Ethical aspects of research in mucosal physiology and disease are challenging, but not insurmountable.
Pristane‐induced arthritis is CD4+ T‐cell dependent
Authors: STASIUK L. M. , GHORAISHIAN M. , ELSON C. J. , THOMPSON S. J.
Published: November 2003

The development of arthritis induced in mice by intraperitoneal injection of the non‐antigenic mineral oil pristane (2,6,10,14‐tetramethylpentadecane) was shown to depend on the presence of CD4+ T cells. Initial experiments assessed the influx of lymphoid cells into the peritoneal cavity of CBA/Igb mice after pristane injection. Both CD4+ and CD8+ cell numbers were maximal around 50 days. Other experiments confirmed our original observation that irradiated pristane‐treated mice failed to develop arthritis unless they were reconstituted with spleen cells from normal donors. This finding has been extended by showing that the population of transferred splenic lymphoid cells must contain CD4+ T cells, while CD8+ T cells and B cells were not required for reconstitution. Conventionally housed and hsp 65‐immunized animals are known to harbour T cells reactive with hsp 65. In addition, hsp 65‐immunized mice are resistant to the development of pristane‐induced arthritis (PIA). Thus, additional experiments assessed the population of splenic T cells activated and proliferating against mycobacterial 65 000 MW heat‐shock protein (hsp 65). In cultures of purified splenic T cells derived from both conventional and hsp 65‐immunized mice, removal of CD4+ T cells significantly reduced the proliferative response to hsp 65, while removal of CD8+ T cells often enhanced the response. These proliferative responses were also shown to be major histocompatability complex (MHC) class II restricted. The present findings demonstrate that PIA is CD4+ T‐cell mediated, and immunodominant environmental antigens such as hsp 65 activate this population of lymphocytes. The CD4+ hsp 65‐reactive population may be pathogenic or protective in PIA, depending upon the route of sensitization.
Vaccination with a multi‐epitopic recombinant allergen induces specific immune deviation via T‐cell anergy
Authors: CAO Y. , YANG M. , LUO Z. , MOHAPATRA S. S.
Published: November 2003

Prophylactic vaccination has recently emerged as a major paradigm toward the prevention and therapy of allergies and asthma; however, the immunological basis of this approach remains to be elucidated. We examined the potential and mechanism of prophylaxis of allergic response in B6D2F1 mice with a multi‐epitopic recombinant allergen, rKBG8·3 (MERA‐8·3), which represents a major group of allergens of grass pollens, used herein as a model of MERA vaccine. Vaccination (subcutaneous) with soluble MERA‐8·3, prior to immunization with the MERA‐8·3 in alum, led to suppression of the IgE antibody response and a concomitant increase in IgG2a antibody response specific to the MERA‐8·3 in a dose‐dependent manner. Analysis of cytokine patterns in spleen and lymph node cells revealed a marked decrease of interleukin‐2 (IL‐2) and IL‐4 production and to a lesser extent a decrease of interferon‐γ (IFN‐γ) synthesis, resulting in an increased ratio of IFN‐γ:IL‐4 in vaccinated–immunized mice compared with untreated–immunized control mice. Furthermore, splenocytes of mice treated with the MERA‐8·3 alone proliferated to MERA‐8·3 in vitro with reduced capacity compared with the splenocytes of MERA‐8·3‐alum immunized mice, owing to a markedly reduced level of IL‐2 production in the former. Collectively, these results suggest that vaccination with the MERA‐8·3 induces T‐cell anergy, which is pivotal to deviation of specific immunity from Th2‐ to Th1‐like, and may serve as an important approach to prevention and therapy of allergic disorders.
Antigen‐presenting cell‐derived signals determine expression levels of CD70 on‘qc primed T cells
Authors: LENS S. M. A. , BAARS P. A. , HOOIBRINK B. , VAN OERS M. H. J. , VAN LIER R. A. W.
Published: November 2003

Interaction between CD27 and its ligand CD70 provides a second signal for T‐cell proliferation and tumour necrosis factor‐α (TNF‐α) production. Whereas CD27 is broadly expressed during T‐cell development, expression of CD70 in vivo is restricted. To determine when CD27–CD70 interactions can occur in immune reactions, we here analysed the regulation of CD70 expression on activated T cells. Mitogenic stimulation of purified T cells with either immobilized CD3 monoclonal antibody (mAb) or a combination of CD2 mAb induces only low levels of CD70 membrane expression. Markedly, expression of the CD27‐ligand is strongly enhanced by antigen‐presenting cells (APC) and APC‐associated signals such as interleukin‐1α (IL‐1α), IL‐12, TNF‐α and CD28‐ligation. In contrast, T‐cell derived cytokines, such as IL‐4, counteract CD70 up‐regulation on activated T cells. Analysis of the small subset of circulating CD70+ T cells revealed that these cells have a primed phenotype as they express CD45RO and HLA‐DR antigens and are in high frequency able to secrete interferon‐γ (IFN‐γ). We conclude that T–T interactions involving CD27 and CD70 are likely to occur relatively early in immune reactions, after productive T‐cell priming by APC and that expression of CD70 on circulating T cells is a reflection of recent priming by antigen.
Localization of IL‐4 and IL‐4 receptors in the human term placenta, decidua and amniochorionic membranes
Authors: DE MORAES‐PINTO M. I. , VINCE G. S. , FLANAGAN B. F. , HART C. A. , JOHNSON P. M.
Published: November 2003

There has been much recent interest in cytokine expression at the materno–fetal interface. Although T‐helper 2 (Th2)‐type cytokines have been described in the murine feto–placental unit, few studies have as yet been performed in human pregnancy. We have examined the production of interleukin‐4 (IL‐4) and expression of IL‐4 receptors in the human term placenta, decidua and amniochorionic membranes. Immunohistochemical analyses revealed that cytotrophoblast, decidual macrophages and both maternal and fetal endothelial cells consistently expressed IL‐4, whereas syncytiotrophoblast and placental macrophages showed an inconsistent pattern between specimens. High‐ and low‐affinity IL‐4 receptors were demonstrated by immunohistochemistry at the same cellular sites as stained for IL‐4, and detection of IL‐4 receptors was also variable in syncytiotrophoblast. Reverse‐transcribed–polymerase chain reaction (RT–PCR) analysis showed that both IL‐4 and its alternative splice variant, IL‐4δ2, are produced both in placental villi and in amniochorionic and decidual tissue. Ligand‐binding assays identified the presence, on isolated term syncytiotrophoblast microvillous plasma membrane vesicle preparations, of functional high‐affinity binding sites for IL‐4 with a Kd in the range 102–112 pm and an apparent receptor density in the rangE 99–102×108 sites/mg protein. Three human choriocarcinoma (BeWo, JEG‐3 and Jar) and one amnion‐derived (AV3) cell lines expressed IL‐4 and both high‐ and low‐affinity IL‐4 receptors. The constitutive expression of both IL‐4 and IL‐4 receptors, together with the novel finding of the alternative splice variant IL‐4δ2 in the immediate tissues at the materno–fetal interface suggest an immunobiological role for IL‐4 in human pregnancy.
T‐cell stimulation and cytokine release induced by staphylococcal enterotoxin A (SEA) and the SEAD227A mutant
Authors: HOLZER U. , ORLIKOWSKY T. , ZEHRER C. , BETHGE W. , DOHLSTEN M. , KALLAND T. , NIETHAMMER D. , DANNECKER G. E.
Published: November 2003

Previous work demonstrated that human cytotoxic T cells activated by superantigens can lyse major histocompatibility complex (MHC) class II‐positive target cells as well as MHC class II‐negative tumour cells coated with conjugates of monoclonal antibodies and superantigens. In order to decrease MHC class II affinity, and therefore unwanted binding of the superantigen staphylococcal enterotoxin A (SEA) to MHC class II molecules, a point mutation was introduced into the SEA gene. This mutation (SEAD227A) resulted in an approximately 3‐log reduction of affinity to human leucocyte antigen (HLA)‐DR, but cytotoxicity mediated by this mutant superantigen towards antibody‐labelled tumour cells is as efficient as cytotoxicity mediated by the native superantigen. We therefore compared the T‐cell activating potency of native and mutated SEA. Our data show that SEAD227A is 4‐ to 5‐log less effective than native SEA when activation of resting T cells is assayed in terms of blast formation, expression of cell surface activation markers and cytokine release. Furthermore, presenting either SEA or SEAD227A to MHC class II‐negative mononuclear cells by MHC class II‐negative tumour cells did not result in significant blast formation of T cells, up‐regulation of CD25 or cytokine release. This suggests that lysis of MHC class II‐negative tumour cells is efficiently induced by monoclonal antibody targeted superantigen, while activation of resting T cells requires additional co‐stimulatory signals.
Effects of 1α,25‐dihydroxyvitamin D3 and cytokines on the expression of MHC antigens, complement receptors and other antigens on human blood monocytes and U937 cells: role in cell differentiation, activation and phagocytosis
Authors: SPITTLER A. , WILLHEIM M. , LEUTMEZER F. , ÖHLER R. , KRUGLUGER W. , REISSNER C. , LUCAS T. , BRODOWICZ T. , ROTH E. , BOLTZ‐NITULESCU G.
Published: November 2003

The effect of calcitriol/1α,25‐dihydroxyvitamin D3, alone and in combination with cytokines, on the expression of various antigens (Ag) on human peripheral blood monocytes and U937 cells was studied by flow cytometry. Both constitutive and interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4), IL‐6 and tumour necrosis factor‐α (TNF‐α)‐induced human leucocyte antigen (HLA)‐DR, HLA‐DP and HLA‐DQ Ag expression on monocytes was significantly down‐regulated by calcitriol, IL‐10 and transforming growth factor‐β (TGF‐β). The effects of calcitriol were concentration dependent and reached maximal inhibitory levels after 3–5 days. Modulation of HLA‐DR by calcitriol and IFN‐γ at the protein level correlated with the amount of mRNA specific for the HLA‐DRα‐chain, as judged by Northern blot analysis. The basal as well as IL‐4, IL‐6, IFN‐γ, TNF‐α and TGF‐β‐driven levels of HLA‐ABC Ag were significantly diminished by calcitriol. On U937 cells calcitriol markedly induced CD11a and CD11b expression and weakly up‐regulated CD11c whereas on monocytes, constitutive CD11a, CD11b and CD11c expression was significantly down‐regulated by calcitriol. The expression of CD14 Ag was strongly induced on U937 cells but only modestly on monocytes. Both the basal level of CD71 and IL‐4, IFN‐γ or TNF‐α‐driven expression was diminished on calcitriol‐treated U937 cells. In addition, calcitriol suppressed the expression of CD71 Ag on monocytes. The ability of monocytes to phagocytize opsonized Escherichia coli was diminished by calcitriol. Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates expression of MHC, CD11b, CD11c, CD14 and CD71 Ag on both monocytes and U937 cells, and impairs the phagocytic property of monocytes.
Constitutive expression of mouse mast cell protease‐1 in normal BALB/c mice and its up‐regulation during intestinal nematode infection
Authors: WASTLING J. M. , SCUDAMORE C. L. , THORNTON E. M. , NEWLANDS G. F. J. , MILLER H. R. P.
Published: November 2003

Rodent intestinal mucosal mast cells (IMMC) store and secrete soluble granule serine proteases, the β‐chymases, which may promote epithelial permeability during intestinal hypersensitivity reactions. The β‐chymase mouse mast cell protease‐1 (mMCP‐1) is generally considered to be expressed late in the in vitro differentiation of mast cells. The purpose of this study was to determine the kinetics of mMCP‐1 transcription and expression in vivo during nematode‐induced IMMC hyperplasia. Concentrations of mMCP‐1 in blood and jejunum of BALB/c mice were quantified by enzyme‐linked immunosorbent assay before and at various stages after infection with the intestinal nematode Nippostrongylus brasiliensis. Mature mMCP‐1 enzyme was detected in jejunal homogenate (194 ng/mg soluble protein) and in blood (8·3 ng/ml serum) from normal uninfected BALB/c mice. Maximal IMMC hyperplasia occurred 7–14 days post infection and was significantly correlated with increased levels of mMCP‐1 in jejunum (r=0·58, P<0·001) and with raised concentrations of mMCP‐1 in serum (r=0·66, P<0·001). Transcription of the mMCP‐1 gene was detected by RNA blotting in normal, uninfected jejunum, but transcription was up‐regulated after infection with maximal transcription occurring on days 7 and 14. In conclusion, mMCP‐1 transcription, storage and secretion occur constitutively in normal BALB/c jejunum but this basal secretion is up‐regulated during nematode infection, suggesting both a physiological and pathological function for this protease.
Relationship between a low toxicity of the mutant A subunit of enterotoxigenic Escherichia coli enterotoxin and its strong adjuvant action
Authors: TSUJI T. , YOKOCHI T. , KAMIYA H. , KAWAMOTO Y. , MIYAMA A. , ASANO Y.
Published: November 2003

In the work described here it was determined if and how unnicking in the A subunit of Escherichia coli enterotoxin at Arg192 or nearby residues affected biological activities of the toxin. The mutant toxin was constructed to lack the nick site in the A subunit by deleting the tripeptide Arg192‐Thr193‐Ile194, which is essential for toxicity. The mutant toxin did not exhibit agmatine ADP‐ribosyltransferase activity in the presence or absence of the ADP‐ribosylation factor and had less diarrhoeal activity and lower induction of cyclic AMP than did LT. The mutant toxin exhibited a much stronger adjuvant action on antibody responses to measles virus, keyhole limpt haemocyanin, bovine immunoglobulin and ovalbumin compared with LT. The altered toxicity of the mutant toxin might be closely related to the potent adjuvant action on antibody responses to antigens. The relationship between two activities is discussed.
Nitric oxide inhibits the secretion of T‐helper 1‐ and T‐helper 2‐associated cytokines in activated human T cells‘pa
Authors: BAUER H. , JUNG T. , TSIKAS D. , STICHTENOTH D. O. , FRÖLICH C. , NEUMANN C.
Published: November 2003

Mechanisms regulating the balance of T‐helper 1 (Th1) and T‐helper 2 (Th2) immune responses are of great interest as they may determine the outcome of allergic and infectious diseases. Recently, in mice, nitric oxide (NO), a powerful modulator of inflammation, has been reported to preferentially down‐regulate Th1‐mediated immune responses. In the present study, we investigated the effect of NO on the production of Th1‐ and Th2‐associated cytokines by activated human T cells and human T‐cell clones. Cytokine secretion was measured in the presence of the NO‐donating agents 3‐morpholinosydnonimine (SIN‐1) and S‐nitroso‐N‐acetylpenicillamine (SNAP). Both NO‐donors markedly inhibited the release of interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), IL‐5, IL‐10 and IL‐4 by anti‐CD3 activated T cells. A preferential inhibition of Th1‐associated cytokines was not observed. Neither was nitrite found in the supernatants of activated T cells, nor was specific mRNA for inducible and constitutive NO synthase detectable, indicating that T cells themselves did not contribute to the observed effect of the NO donors. Costimulation with anti‐CD28 monoclonal antibodies (mAb) prevented SIN‐1/SNAP‐mediated down‐regulation of cytokine production only in part. In contrast, when T cells were stimulated by phorbol‐ester and ionomycin, they were refractory to SIN‐1‐induced inhibition of cytokine production. When SIN‐1 was added after the onset of anti‐CD3 stimulation, the inhibitory effect was found to be less pronounced, indicating that SIN‐1 may interfere with early signal transduction events. The addition of superoxide dismutase (SOD) and catalase did not restore the effects of SIN‐1, demonstrating that the inhibition of cytokines was due to NO and not to oxygen intermediates. Furthermore, 8‐Br‐cGMP‐mediated increase of intracellular cGMP caused the same pattern of cytokine inhibition as observed with SIN‐1 and SNAP. Using a single cell assay, these agents were shown to reduce the frequency of IFN‐γ‐producing T cells, suggesting that not all T cells are susceptible to SIN‐1/SNAP. However, cytokine production by purified T‐cell subpopulations (CD4+, CD8+, CD45RA+, and CD45RO+) was equally impaired by NO donors. In conclusion, in contrast to the murine system, our results do not provide evidence that NO preferentially inhibits Th1‐cytokine secretion of activated human T cells in vitro.
Accumulation of CTLA‐4 expressing T lymphocytes in the germinal centres of human lymphoid tissues‘pa
Authors: CASTAN J. , TENNER‐RACZ K. , RACZ P. , FLEISCHER B. , BRÖKER B. M.
Published: November 2003

CTLA‐4, a coreceptor with sequence homology to CD28 is expressed on T cells after activation. Mice deficient for CTLA‐4 die young from massive infiltration of many organs by activated T cells, which highlights the essential inhibitory role the coreceptor plays in the regulation of the immune response. To study the prevalence and distribution of CTLA‐4 in situ immunohistological analyses were carried out on human tonsils and lymph nodes. Expression of CTLA‐4 was restricted to αβ T cells, and CTLA‐4+ B cells were not observed. In T‐cell areas, 2–10% of T cells were positive for CTLA‐4 with similar percentages in the CD4+ and CD8+ subpopulations. In the germinal centres (GC) the fraction of CTLA‐4+ T cells was much higher (70–90%). This was due to frequent expression of CTLA‐4 on the CD4+ helper subpopulation. GC CD8+ T cells were rare and mostly did not express the coreceptor. The CTLA‐4+ T‐cell fraction was also over‐represented among intraepithelial tonsillar T cells. Cycling (Ki‐67+) and apoptotic (TUNEL+) T cells were never positive for CTLA‐4, while a subset of CD25+ cells did express the coreceptor. Since CTLA‐4 is essential for the physiological limitation of the immune response, GC T cells, which are mostly CTLA‐4 positive, might be important in this process.