bluuebunny/crossref_metadata_2025 huggingface dataset:
Different endothelins stimulate cytokine production by peritoneal macrophages and microglial cell line
Authors: Speciale  , Roda  , Saresella  , Taramelli  , Ferrante
Published: March 2003

Endothelins (ETs), potent vasoconstricting peptides, are produced by macrophages upon stimulation and may participate in the amplification or regulation of the inflammatory response. However, it is not clear whether ETs can act in an autocrine manner on macrophages and which role they play in relationship with other cytokines. To address these issues, we studied the effects of ETs on the production of inflammatory cytokines by mouse peritoneal macrophages or by a retrovirus‐transformed microglial cell line. Here, we report that ET‐2, but not ET‐1 or ET‐3, is able to stimulate the production of interleukin‐1 (IL‐1) and interleukin‐6 (IL‐6) by peptone‐elicited mouse macrophages (pMO). In contrast, ET‐3 and ET‐1, but not ET‐2, are active on microglial cells. No tumour necrosis factor‐α (TNF‐α) or nitric oxide (NO) were detected in the supernatants of ET‐stimulated cultures. The activity of ET‐2 on pMO was time and dose dependent and was inhibited by the addition of ETA and ETB receptor antagonists, BQ123 and IRL1038, respectively. In addition, when pMO were stimulated by interferon‐γ (IFN‐γ) in the presence of ET‐2, a significant inhibition of IL‐6 and IL‐1 production was observed compared with the effects of the same doses of IFN‐γ or ET‐2 used separately. The inhibition was specifically due to the activity of ET‐2, since it was reversed by the addition of BQ123 or IRL1038. Similar results were seen when the content of NO in the supernatants of pMO stimulated by IFN‐γ plus ET‐2 was evaluated. These results suggest that ETs may possess both a pro‐inflammatory action on macrophages from different tissues and a regulatory activity on IFN‐γ.
Neither interleukin‐6 nor signalling via tumour necrosis factor receptor‐1 contribute to the adjuvant activity of Alum and Freund’s adjuvant
Authors: Brewer  , Conacher  , Gaffney  , Douglas  , Bluethmann  , Alexander
Published: March 2003

The potential contribution made by the inflammatory cytokines, interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α) to the adjuvant activity of aluminium hydroxide gels (Alum) or Freund’s complete adjuvant (FCA) was studied by comparing the immunological responses of IL‐6‐ or TNF receptor 1‐ (p55; TNFR‐1) deficient mice with immunocompetent control mice. While both TNFR‐1‐ and IL‐6‐deficient mice primed with ovalbumin (OVA) prepared in either Alum or FCA produced similar IgG1 responses in comparison to control mice, the pattern of T‐helper type 1‐ (Th1) dependent IgG2a production was significantly altered. In TNFR‐1‐deficient mice, IgG2a responses were greater than in control mice when FCA, but not when Alum, was used as an adjuvant. Correspondingly, spleen cells from FCA‐inoculated TNFR‐1‐deficient mice restimulated with antigen in vitro produced higher Th1 cytokine (interferon‐γ; IFN‐γ) levels with no alteration in Th2 cytokine (IL‐4, IL‐5, IL‐6 and IL‐10) production in comparison with wild‐type mice. Higher levels of IgG2a were also detected in IL‐6‐deficient mice compared with wild‐type mice following inoculation with OVA prepared in either FCA or in Alum. Furthermore, analysis of cytokine production by spleen cells revealed that both Th1 and Th2 cytokine production was higher in IL‐6‐deficient mice compared with control mice. As the majority of the effects of TNF‐α are mediated via TNFR‐1, we conclude that this cytokine inhibits the adjuvant activities of FCA. Furthermore, the results also imply that immunopotentiating effects of FCA or Alum adjuvant are both inhibited by IL‐6.
Predominant recognition of species‐specific determinants of the GroES homologues from Mycobacterium leprae and M. tuberculosis
Authors: Chua‐Intra  , Ivanyi  , Hills  , Thole  , Moreno  , Vordermeier
Published: March 2003

The Mycobacterium leprae and M. tuberculosis 10 000 MW heat‐shock protein homologues of GroES have previously been identified as major immunogens for human T cells. We used synthetic peptides to characterize the determinants recognized by murine T cells. The findings suggest that, despite 90% sequence identity between these two proteins, T cells recognize prominently the species‐specific determinants localized within amino acid residues 21–40 and 49–72. Analysis of the molecular determinants of species‐specificity for the M. leprae GroES sequence 25–40, using T‐cell hybridomas and major histocompatibility complex (MHC)‐binding assays, led to the identification of epitope cores and critical residues. Interestingly, closely overlapping epitope cores were found to be restricted by either H‐2Ad (24–34) or H‐2Ed (28–34). Furthermore, the site recognized by the M. leprae‐specific monoclonal antibodies ML06 and ML10 was also localized in the overlapping sequences 25–31 and 25–29. In conclusion, we demonstrated that immunodominant species‐specific T‐ and B‐cell epitopes can be found in a mycobacterial heat‐shock protein despite its highly conserved amino acid sequence. This finding suggests the feasibility of identifying a sufficient number of M. leprae‐specific determinants for a composite T‐cell immunodiagnostic reagent for tuberculoid leprosy.
Distinct T‐cell subtypes induced with whole cell and acellular pertussis vaccines in children
Authors: Ryan  , Murphy  , Ryan  , Nilsson  , Shackley  , Gothefors  , Øymar  , Miller  , Storsaeter  , Mills
Published: March 2003

Recent clinical trials have demonstrated that new generation acellular pertussis vaccines can confer protection against whooping cough. However, the mechanism of protective immunity against Bordetella pertussis infection induced by vaccination remains to be defined. We have examined cellular immune responses in children immunized with a range of acellular and whole cell pertussis vaccines. Immunization of children with a potent whole‐cell vaccine induced B. pertussis‐specific T cells that secreted interferon‐γ (IFN‐γ), but not interleukin‐5 (IL‐5). In contrast, T cells from children immunized with acellular pertussis vaccines secreted IFN‐γ and/or IL‐5 following stimulation with B. pertussis antigens in vitro. These observations suggest that protective immunity conferred by whole‐cell vaccines, like natural immunity, is mediated by type 1 T cells, whereas the mechanism of immune protection generated with acellular vaccines may be more heterogenous, involving T cells that secreted type 1 and type 2 cytokines.
The dual role of interferon‐γ in experimental Staphylococcus aureus septicaemia versus arthritis
Authors: Zhao  , Nilsson  , Tarkowski
Published: March 2003

To evaluate the role of interferon‐γ (IFN‐γ) in Staphylococcus aureus infection, we investigated the effects of supplementation with and neutralization of IFN‐γ during septicaemia and arthritis in a murine model. In vivo administration of IFN‐γ both before and after bacterial inoculation significantly decreased mortality on one hand but enhanced the development of arthritis on the other. Treatment of mice with anti‐IFN‐γ monoclonal antibodies (mAb) before and after bacterial inoculation did not significantly influence the survival rate but decreased the frequency and severity of arthritis. The beneficial effect of supplementation with IFN‐γ on septicaemia was correlated to the increased phagocytosis and bacterial clearance from liver and kidneys. The down‐regulation of the development of arthritis by anti‐IFN‐γ mAb was accompanied by the decreased serum tumour necrosis factor‐α, interleukin‐6 and interleukin‐1β levels. These results demonstrate a significant role for IFN‐γ in simultaneous protection against septicaemia but promotion for the development of septic arthritis.
In vivo exposure to Porphyromonas gingivalis up‐regulates nitric oxide but suppresses tumour necrosis factor‐α production by cultured macrophages
Authors: FROLOV  , HOURI‐HADAD  , SOSKOLNE  , SHAPIRA
Published: March 2003

The present study was designed to test whether the functional response of mouse macrophages elicited by chronic exposure to bacteria will be different from that of cells elicited by a non‐bacterial irritant. Macrophage elicitation was conducted by Porphyromonas gingivalis, a major periodontal pathogen, in comparison to a standard elicitation by thioglycollate (TG). We measured lipopolysaccharide (LPS)‐induced nitric oxide (NO) and tumour necrosis factor‐α (TNF‐α) secretion by the elicited macrophages, and the expression of inflammatory cytokines in the whole elicited cell population. In addition, we tested the response of TG‐elicited macrophages to pretreatment with P. gingivalis LPS in vitro. Mouse peritoneal macrophages were harvested 4 days after intraperitoneal injection of TG or heat‐killed P. gingivalis. TG‐elicited macrophages produced undetectable levels of TNF‐α and approximately 0·5 μm of NO. The stimulation of the macrophages with LPS resulted in the secretion of NO and TNF‐α in a dose‐dependent manner. The P. gingivalis‐elicited macrophages produced basal levels of approximately 5 μm NO, but TNF‐α was not detectable. LPS stimulation of these cells further increased the secretion of NO eightfold while TNF‐α remained undetectable. The NO secretion by P. gingivalis‐elicited cells was significantly higher than that by TG‐elicited cells. Examination of cytokine expression in the whole elicited cell population revealed that both P. gingivalis‐elicited cells and TG‐elicited cells expressed messenger RNA for interleukin‐2 (IL‐2), TNF‐α and interferon‐γ (IFN‐γ), but not for IL‐4. IL‐6 was expressed in P. gingivalis‐elicited cells only. Pretreatment of TG‐elicited macrophages with P. gingivalis LPS for 24 hr prior to a second LPS challenge resulted in down‐regulation of TNF‐α secretion and up‐regulation of NO secretion, a response similar to that seen in P.gingivalis‐elicited peritoneal macrophages. The results suggest that the in vivo exposure of resident macrophages to P. gingivalis induces functional changes in peritoneal macrophages. These changes might be due to the effect of P. gingivalis LPS.
Activation‐dependent expression of the insulin‐like growth factor binding protein‐2 in human lymphocytes
Authors: Föll J. L. , Dannecker L. , Zehrer C. , Hettmer S. , Berger J. , Elmlinger M. , Niethammer D. , Ranke M. B. , Dannecker G. E.
Published: March 2003

The expression of the insulin‐like growth factor binding protein‐2 (IGFBP‐2) was assayed in mononuclear cells originating from different organs of the immune system. All mononuclear cells studied did express IGFBP‐2, but the expression level was found to be dependent on the cell type and origin of the cell. T cells showed a higher expression of IGFBP‐2 mRNA than did B cells, and CD34+stem cells expressed IGFBP‐2 mRNA at a high level. Expression was highest in bone marrow and thymus. Stimulation of peripheral mononuclear cells resulted in a marked increase of IGFBP‐2 mRNA and also intracellular IGFBP‐2, as analysed by fluorescence staining. This increase parallels the increase of other known T‐cell activation markers. Furthermore, the increase of intracellular IGFBP‐2 seems to precede T‐cell blast formation and all T cells in active phases of the cell cycle have high levels of IGFBP‐2. Our results provide a basis for further investigations on the contribution of the IGF‐system to the regulation of T‐cell proliferation and differentiation. IGFBP‐2, in particular, may have an important influence in the regulation of T‐cell activation and proliferation.

A 23187, calcium ionophore IGF I/II, insulin‐like growth factor‐I/II IGFBPs, insulin‐like growth factor binding proteins PBMC, peripheral blood mononuclear cell MNC, mononuclear cell MACS, magnetic‐activated cell sorting GAPDH, glycerinaldehydphosphate RT‐PCR, reverse transcriptase–polymerase chain reaction.
Intranasal administration of human immunodeficiency virus type‐1 (HIV‐1) DNA vaccine with interleukin‐2 expression plasmid enhances cell‐mediated immunity against HIV‐1
Authors: XIN K.‐Q. , HAMAJIMA K. , SASAKI S. , HONSHO A. , TSUJI T. , ISHII N. , CAO X‐R. , LU Y. , FUKUSHIMA J. , SHAPSHAK P. , KAWAMOTO S. , OKUDA K.
Published: March 2003

DNA vaccine against human immunodeficiency virus type‐1 (HIV‐1) can induce substantial levels of HIV‐1‐specific humoral and cell‐mediated immunity. To develop more potent HIV‐1 DNA vaccine formulations, we used a murine model to explore the immunomodulatory effects of an interleukin‐2 (IL‐2) expression plasmid on an HIV‐1 DNA vaccine following intranasal administration of the combination. When the vaccine and expression plasmid were incorporated into cationic liposomes and administered to mice, the HIV‐1‐specific delayed‐type hypersensitivity response and cytotoxic T lymphocyte activity were significantly increased. Restimulated immune lymphoid cells showed enhanced production of both IL‐2 and interferon‐γ and reduced secretion of IL‐4. The level of total antibody to HIV‐1 antigen was not greatly changed by coadministration of the DNA vaccine and IL‐2 expression plasmid. An analysis of serum HIV‐1‐specific IgG subclasses showed a significant drop in the IgG1/IgG2a ratio in the group that received the plasmid–vaccine combination. These results demonstrate that the IL‐2 expression plasmid strongly enhances the HIV‐1‐specific immune response via activation of T helper type‐1 cells.
Localization in situ of costimulatory molecules and cytokines in B‐cell non‐Hodgkin’s lymphoma
Authors: VYTH‐DREESE  , BOOT  , DELLEMIJN  , MAJOOR  , OOMEN  , LAMAN  , VAN MEURS  , DE WEGER  , DE JONG
Published: March 2003

Costimulatory molecules are essential in cognate interactions between T and B lymphocytes. To study the prerequisites of functional interactions between malignant B cells and intermingled T cells in B‐cell non‐Hodgkin’s lymphomas (B‐NHL), we examined the expression of CD40, CD80 and CD86 and their ligands CD40 ligand (CD40L, CD154), CD28 and CTLA4 (CD152) using immunohistochemistry and confocal laser scanning microscopy. Almost all mucosa‐associated lymphoid tissue (MALT) NHL were positive for CD40 and CD80 and in nine out of 14 cases were positive for CD86. The majority of follicle centre cell lymphomas (FCCL) expressed CD40, but were heterogeneous in their expression of CD80 and CD86. Most diffuse large cell lymphomas (DLCL) were CD80+, but lacked expression of CD86. These patterns reflect the differences in phenotype of normal marginal‐zone B cells (as counterparts of MALT NHL) and germinal centre cells (as counterparts of FCCL and DLCL). Counter‐receptors on T cells were detectable in 13 of 14 MALT NHL, 12 of 16 FCCL but only occasionally in DLCL (three of 12 cases). A subgroup of FCCL was identified with T‐cell expression of CD40L, CD28 and CTLA4 simultaneously with strong expression of CD40 and CD86 on the tumour B cells. These results indicate that MALT NHL and a subset of FCCL are most optimally equipped for functional interactions with T cells. This may be supported by the demonstration of cytokine production – mainly in T cells – in MALT NHL [interleukin‐2 (IL‐2), interferon‐γ (IFN‐γ), IL‐10] and FCCL (IL‐2, IFN‐γ) and to a lesser extent in DLCL.
Enhancement of activation‐induced cell death by fibronectin in murine CD4+ CD8+ thymocytes
Authors: TAKAYAMA  , KINA  , KATSURA  , TADAKUMA
Published: March 2003

extracellular matrix molecule in the thymus, in the cell death induced by activation via the T‐cell antigen receptor. FN alone did not increase cell death in murine thymocytes above the baseline level, but it significantly enhanced the cell death induced by fixed anti‐CD3 monoclonal antibody (mAb), especially when a high concentration of anti‐CD3 mAb was used. DNA fragmentation increased in parallel with cell death, indicating that cell death was a result of the apoptosis. Fluorescence‐activated cell sorter (FACS) analysis revealed that the activation‐induced cell death (AICD) caused by anti‐CD3 mAb alone, or by a combination of anti‐CD3 mAb and FN, occurred selectively in CD4+ CD8+ thymocytes. Very late activation antigen (VLA)‐4 and VLA‐5 are two major ligands to FN on thymocytes. The expression of both ligands was investigated at different stages of thymocyte development. VLA‐4 was predominantly expressed at the CD4− CD8− stage, and thereafter the expression was reduced, whereas VLA‐5 was constantly expressed during maturation. Furthermore, the enhancing effect by FN was inhibited in the presence of the Gly‐Arg‐Gly‐Asp‐Ser‐Pro (GRGDSP) peptide but not in the presence of the connecting segment‐1 (CS‐1) peptide, suggesting that enhancement of AICD observed in CD4+ CD8+ thymocytes is mediated through VLA‐5.
Regulatory effects of Th1‐type (IFN‐γ, IL‐12) and Th2‐type cytokines (IL‐10, IL‐13) on parasite‐specific cellular responsiveness in Onchocerca volvulus‐infected humans and exposed endemic controls
Authors: Soboslay  , Lüder  , Riesch  , Geiger  , Banla  , Batchassi  , Stadler  , Schulz‐Key
Published: March 2003

The present study investigated in vitro the regulatory effects of T helper 1 (Th1)‐type (interferon‐γ, IFN‐γ; interleukin‐12, IL‐12) and Th2‐type cytokines (IL‐10, IL‐13) on Onchocerca volvulus‐specific cellular reactivity in onchocerciasis patients, and in exposed endemic control individuals presenting no clinical and parasitological signs of disease. In both patients and controls, addition of IL‐10 dose‐dependently depressed O. volvulus antigen (OvAg)‐specific cellular proliferation, and peripheral blood mononuclear cells (PBMC) from patients who were more sensitive to the suppressive effect of IL‐10 than those from endemic controls. However, neutralization of IL‐10 by specific antibody did not reverse cellular hyporesponsiveness. In contrast to the inhibitory effects of IL‐10, exogenous IL‐12 and IL‐13 augmented PBMC proliferative responses to OvAg both in patients and controls (P<0·01) and neutralizing of IL‐12 or IL‐13 significantly decreased OvAg‐specific proliferation in both groups. Exogenous IFN‐γ did not activate OvAg‐specific proliferative responses in patients, but anti‐IFN‐γ antibodies abolished cellular reactivity to OvAg. Antibody to IL‐10 increased (P<0·05) OvAg‐specific production of IL‐5, IL‐12 and IFN‐γ, and inversely, anti‐IFN‐γ enhanced IL‐10 (in patients only) and IL‐5 and IL‐13 in both patients and controls. Neutralization of IL‐12 activated OvAg‐specific production of IL‐10, IL‐2 and IFN‐γ. In conclusion, despite of an overproduction of IL‐10, which suppressed cellular reactivity in patients and control individuals, OvAg‐specific cellular responses were activated in vitro by exogenous supplementation with IL‐12 and IL‐13, and cytokine neutralization experiments confirmed that distinct type 1 and type 2 T helper cytokines cross‐regulate expression and magnitude of O. volvulus‐specific cellular responsiveness in humans.
Differential effects of anti‐rat CD11b monoclonal antibodies on granulocyte adhesiveness
Authors: DRAŠKOVIĆ‐PAVLOVIĆ  , VAN DER LAAN  , PEJNOVIĆ  , DIJKSTRA  , ČOLIĆ
Published: March 2003

Four different monoclonal antibodies (mAbs) reactive with rat CD11b (ED7, ED8, OX‐42 and 1B6c) have been characterized for their ability to induce homotypic aggregation of granulocytes or to modify granulocyte adhesiveness triggered by phorbol myristate acetate (PMA) or N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP). Cross‐blocking experiments showed that these mAbs recognize at least three different epitopes on CD11b. OX‐42 mAb recognizes an inhibitory epitope since the mAb inhibited homotypic aggregation of granulocytes and their adherence to plastic in the presence of PMA or fMLP. ED7 and ED8 induced homotypic aggregation of granulocytes which was blocked by OX‐42 and anti‐CD18 mAb (WT3) suggesting that CR3 itself is involved in the adhesion process. The aggregation was dependent on active cell metabolism, intact cytoskeleton, divalent cations and activation of tyrosine kinases sensitive to genistein. Staurosporine, okadaic acid and orthovanadate potentiated the aggregation. ED7 and ED8 potentiated homotypic aggregation and adhesion of granulocytes to plastic caused by fMLP, but inhibited granulocyte adhesion to plastic induced by PMA. 1B6c recognizes an epitope that transmits a proaggregatory signal upon binding of the mAb but only if the granulocytes are in contact with plastic or are activated by fMLP. In contrast, 1B6c inhibited granulocyte adhesion to plastic triggered by PMA or fMLP. These data suggest the existence of functionally different epitopes on rat CD11b and indicate that some anti‐CD11b mAbs are able to functionally activate CR3.
High level expression of Nramp1G169 in RAW264.7 cell transfectants: analysis of intracellular iron transport
Authors: ATKINSON  , BARTON
Published: March 2003

Nramp1 (natural resistance‐associated macrophage protein) was positionally cloned as the defective biallelic locus in inbred mouse strains associated with uncontrolled proliferation of obligate intracellular macrophage pathogens. The causative defect was described as G169D within membrane spanning domain 4 of a transporter. The biochemical activity of Nramp1 is implied from sequence conservation with Nramp2. Nramp2 encodes a divalent cation transporter and is the carrier of a defect in models of microcytic anaemia, associated with impaired intestinal iron uptake. Iron sequestration has been proposed as an antimicrobial mechanism. Therefore, such an activity for Nramp1 is consistent with model systems. Here we showed that Nramp1 directs iron transport within the macrophage. We describe stable, high‐level Nramp1G169 allele‐derived polypeptide expression in Balb/c Nramp1D169 RAW264.7 cells. Transfectants express levels, comparable to those in Nramp1G169‐resistant macrophages, of a 90–100×103 MW Nramp1 polypeptide. Expression of the Nramp1 polypeptide correlates with lower cellular iron loads and a reduced chelatable iron pool following challenge with iron: nitrilotriacetate. Pulse chase experiments support an enhanced iron flux in expressing cells. These data are supported using the fluorescent iron probe calcein. In Nramp1G169‐expressing cells we observed an increased iron flux into the cytoplasm from a calcein‐inaccessible cellular location. These data suggest Nramp1, in resting macrophage cells, mobilizes iron, from an intracellular vesicle, which is destined for cell secretion. We propose that under these conditions Nramp1 plays a role in a salvage pathway of iron recycling.
Synergistic action of stem‐cell factor and interleukin‐7 in a human immature T‐cell line
Authors: KEES  , FORD
Published: March 2003

The thymus provides the microenvironment that is optimal for T‐cell differentiation. The most immature cells in the human thymus express the stem‐cell marker CD34 and they respond to cytokines, including stem‐cell factor (SCF) and interleukin‐7 (IL‐7). For the normal progression of T‐cell development these two cytokines appear to be vital. We have established and characterized a human pre‐T‐cell line, PER‐487, which mirrors this requirement. This study shows that the simultaneous presence of IL‐7 and SCF produces a proliferative response far exceeding additive effects. Furthermore, providing these signals in succession did not achieve the effect observed when they were provided simultaneously. This finding suggests that the effect was not mediated via secretion of molecules or modulation of surface expression. The convergence of the signal transduction pathways of the two cytokines is not known, thus cell line PER‐487 provides a unique model for studying the synergistic interaction of IL‐7 and SCF.
Protein‐kinase‐specific inhibitors block Langerhans’ cell migration by inhibiting interleukin‐1α release
Authors: SHANKAR  , JOHNSON  , KUSCHEL  , RICHINS  , BURNHAM
Published: March 2003

Previous studies have shown that depletion of Langerhans’ cells (LC) from murine epidermis by the superantigen, staphylococcal enterotoxin A (SEA) involves interleukin‐1α (IL‐1α) and is inhibitable by agents that block G‐protein‐associated kinases. The purpose of this study was to determine whether specific kinase inhibitors block LC depletion by inhibiting IL‐1α release and to ascertain whether LC depletion by SEA involves cell migration. These goals were addressed by measuring the IL‐1α release within whole or LC‐depleted epidermal cell suspensions in the presence of SEA and/or H‐7 (an inhibitor of protein kinase C) or H‐8 (an inhibitor of G‐protein‐associated kinases) and by examining the migration of cells with LC markers in SEA‐treated skin sections. The results suggest that LC depletion by SEA involves migration and that this migration is blocked by protein kinase inhibitors, at least in part, through inhibition of SEA‐induced IL‐1α release by epidermal cells.
Successive tick infestations selectively promote a T‐helper 2 cytokine profile in mice
Authors: FERREIRA  , SILVA
Published: March 2003

Several studies have revealed that T lymphocytes and cytokines play a crucial role in determining the outcome of parasitic infections in terms of protective immunity. In this study we found that Rhipicephalus sanguineus tick saliva stimulates transforming growth factor‐β (TGF‐β), and reduces interleukin‐12 (IL‐12) secretion by cells from normal C3H/HeJ mice. Moreover, murine lymph node cells harvested 6 days after the fourth infestation with ticks presented an 82·4% decrease in their proliferative response to concanavalin A (Con A) compared with the response of control cells. In addition, lymph node cells cultured in the presence of Con A showed a T‐helper 2‐type (Th2‐type) cytokine profile, represented by augmented IL‐4 and IL‐10 and TGF‐β. On the other hand, the IL‐2, interferon‐γ (IFN‐γ) and IL‐12 synthesis was significantly inhibited. These results indicate that ticks may modulate the host’s immune response through saliva injection. Consideringthat C3H/HeJ mice develop no protective immunity to R. sanguineus infestation, our results suggest that tick‐induced Th2‐type cytokines and a decreased proliferative response probably lead the host to a susceptible state to both tick and tick‐transmitted pathogens.
Differentiation‐dependent switch in protein kinase C isoenzyme activation by FcγRI, the human high‐affinity receptor for immunoglobulin G
Authors: MELENDEZ  , HARNETT  , ALLEN
Published: March 2003

Aggregation of receptors for the constant region (Fc) of immunoglobulin G on myeloid cells results in endocytosis or phagocytosis and cellular activation. Previous work has shown, using the cell line U937, that the high‐affinity immunoglobulin G receptor, FcγRI, activates alternate intracellular signalling pathways depending on the cell differentiation state, which results in a marked change in the nature of calcium transients within the cell. Here, we show that protein kinase C (PKC) is activated in both interferon‐γ (IFN‐γ) ‐primed and dibutyryl cyclic AMP (dbcAMP) ‐differentiated cells but that the nature of the particular isoenzymes recruited differs. Thus, in IFN‐γ‐primed U937 cells, FcγRI aggregation results in an increase of PKC activity which is essentially calcium independent resulting from the translocation to the membrane of the novel PKCs, δ and ε, together with the atypical PKC ζ. However, in cells differentiated to a more macrophage phenotype, all PKC enzyme activity after receptor aggregation is calcium dependent. Consistent with this finding, the isoenzymes translocated to the nuclear‐free membrane fraction are the conventional PKCs α, β and γ; results consistent with our previous finding that FcγRI couples to phospholipase C in such dbcAMP‐differentiated cells. Thus, the nature of PKC isoenzyme activated following FcγRI aggregation is defined by differentiation. The calcium dependence of the PKC isoenzyme is consistent with the duration of calcium transients previously reported in the two differentiation states.
Effect of CD8+ T‐cell depletion on bronchial hyper‐responsiveness and inflammation in sensitized and allergen‐exposed Brown–Norway rats
Authors: HUANG  , MACARY  , KEMENY  , CHUNG
Published: March 2003

We examined the role of CD8+ T cells in a Brown–Norway rat model of asthma, using a monoclonal antibody to deplete CD8+ T cells. Ovalbumin (OA)‐sensitized animals were given anti‐CD8 antibody (0·5 mg/rat) intravenously 1 week prior to exposure to 1% OA aerosol and were studied 18–24 hr after aerosol exposure. Following administration of anti‐CD8 antibody, CD8+ cells were reduced to <1% of total lymphocytes in whole blood and in spleen. In sensitized animals, OA exposure induced bronchial hyper‐responsiveness (BHR), accumulation of eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid, and also an increase in tissue eosinophils and CD2+, CD4+ and CD8+ T cells in airways. Anti‐CD8 antibody caused a further increase in allergen‐induced BHR (P<0·03, compared with sham‐treated animals), together with a significant increase in eosinophil number in BAL fluid (P<0·05). While CD2+ and CD4+ T cells in airways were not affected by anti‐CD8 treatment, the level of CD8+ T cells was significantly reduced in sensitized, saline‐exposed animals (P<0·04, compared with sham‐treated rats), and sensitized and OA‐challenged rats (P<0·002, compared with sham‐treated rats). Using reverse transcription–polymerase chain reaction, an increase of T helper (Th)2 cytokine [interleukin (IL)‐4 and IL‐5], and also of Th1 cytokine [interferon‐γ (IFN‐γ) and IL‐2], mRNA in the lung of sensitized and OA‐exposed animals was found; after CD8+ T‐cell depletion, Th1 cytokine expression was significantly reduced (P<0·02), while Th2 cytokine expression was unchanged. CD8+ T cells have a protective role in allergen‐induced BHR and eosinophilic inflammation, probably through activation of the Th1 cytokine response.
Interleukin‐15 production at the early stage after oral infection with Listeria monocytogenes in mice
Authors: MITANI  , NISHIMURA  , HIROSE  , WASHIZU  , KIMURA  , TANAKA  , YAMAMOTO  , NOGUCHI  , YOSHIKAI
Published: March 2003

We previously reported that exogenous interleukin‐15 (IL‐15) induces proliferation and activation of intestinal intraepithelial lymphocytes (i‐IEL) in naive mice. To investigate the ability of endogenous IL‐15 to stimulate i‐IEL in vivo, we monitored i‐IEL and intestinal epithelial cells (i‐EC) in mice after an oral infection with Listeria monocytogenes. Although the populations of αβ and γδ i‐IEL were not significantly changed after the oral infection, the expression level of interferon‐γ (IFN‐γ) was increased both at transcriptional and protein levels, and a conversely marked decrease in interleukin‐4 (IL‐4) was detected in the i‐IEL on day 1 after infection as compared with before infection. The T helper 1 (Th1)‐biased response of i‐IEL coincided with a peak response of IL‐15 production in the i‐EC after oral infection. These results suggested that IL‐15 produced from i‐EC may be at least partly involved in the stimulation of i‐IEL to produce IFN‐γ after oral infection with L. monocytogenes.
Mammalian granulocyte–macrophage colony‐stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish
Authors: KANELLOS  , SYLVESTER  , BUTLER  , AMBALI  , PARTIDOS  , HAMBLIN  , RUSSELL
Published: March 2003

A eukaryotic plasmid DNA carrying the AACGTT CpG motif in its ampR gene is a ‘danger’ signal for mice and caused an increase in the specific antibody titres of fish and mice after immunization with β‐galactosidase (β‐gal). A second pUC‐based plasmid, which is inactive in mice and contains the GACGTC CpG motif in its cytomegalovirus (CMV) promoter, had no effect on antibody responses to β‐gal in either fish or mice. A synthetic oligonucleotide, which contains the GACGTT motif, potentiated antibody responses to co‐administered β‐gal protein in mice, but not in fish. This is early evidence that lower and higher vertebrates recognize different unmethylated CpG motifs as ‘danger’ signals. In addition, plasmid DNA expressing mouse granulocyte–macrophage colony‐stimulating factor (GM‐CSF) had a marked effect on cytotoxic T‐cell‐like activity in fish by reducing the average number of myofibres that expressed β‐gal, 28 days after co‐injection with plasmid DNA expressing β‐gal. Although the mechanism by which the mouse GM‐CSF exerted its biological effects in fish is unknown, this finding might have important implications for fish vaccination, particularly when cytotoxic T cells may play a critical role.
Enterobacterial infection modulates major histocompatibility complex class I expression on mononuclear cells
Authors: Kirveskari  , He  , Leirisalo‐Repo  , Mäki‐Ikola  , Wuorela  , Putto‐Laurila  , Granfors
Published: March 2003

Major histocompatibility complex (MHC) class I expression is reduced in several viral infections, but it is not known whether the same happens during infections caused by intracellular enterobacteria. In this study, the expression of MHC class I antigens on peripheral blood mononuclear cells (PBMC) from 16 patients with Salmonella, Yersinia, or Klebsiella infection was investigated. During or after the acute infection, the expression of MHC class I antigens was markedly decreased in eight patients, all with genotype HLA‐B27, and six out of eight with reactive arthritis (ReA). A significant decrease of monomorphic MHC class I was found in three patients, of HLA‐B27 in eight (P<0·05) and of HLA‐A2 in two. However, patients negative for the HLA‐B27 genotype, or healthy HLA‐B27‐positive individuals, did not have a significant decrease of MHC class I antigens. During the decreased expression on the cell surface, intracellular retention of MHC class I antigens was observed, whereas HLA‐B27 mRNA levels did not vary significantly. This is the first evidence that enterobacterial infection may down‐regulate expression of MHC class I molecules in vivo and that down‐regulation is predominant in patients with the HLA‐B27 genotype.
Kinetics of interferon‐γ secretion and its regulatory factors in the early phase of acute graft‐versus‐host disease
Authors: Hu H. Z. , Li G. L. , Lim Y. K. , Chan S. H. , Yap E. H.
Published: March 2003

Increased serum levels of interferon‐γ (IFN‐γ) have been observed in acute graft‐versus‐host disease (GVHD). Recent in vitro studies have demonstrated that interleukin‐12 (IL‐12) and interleukin‐18 (IL‐18) synergistically up‐regulate IFN‐γ secretion. In this communication, we investigated the factors relevant to IFN‐γ secretion in acute GVHD. A murine model of acute GVHD was established by injecting donor spleen cells into severe combined immunodeficiency (SCID) mice. A series of specimens, including sera, livers and spleens derived from the GVHD mice, were investigated with histological examination, enzyme‐linked immunosorbent assay (ELISA), flow cytometry, and semiquantitative reverse transcription–polymerase chain reaction (RT–PCR). IFN‐γ secretion increased in serum 3 days after spleen cell transfer, peaked on day 7, and then gradually decreased close to the baseline level by day 35. A synchronized increase of activated T cells and mRNA expression of IL‐12, IL‐18 and their respective receptors was observed after spleen cell transfer. However, only the kinetic expression pattern of IL‐12 receptor (IL‐12R) β2 chains was closely correlated with that of IFN‐γ, while IL‐12 dropped to the baseline level earlier than IFN‐γ. Therefore, IFN‐γ expression in the early phase of acute GVHD is a mono‐peak and self‐restricted pattern. Its secretion is closely related with T‐cell activation, the presence of IL‐12, IL‐18 and their respective receptors. However, the limiting factors for IFN‐γ secretion seem to be IL‐12 and IL‐12R β2 chains.
Morphine‐induced macrophage apoptosis: the role of transforming growth factor‐β
Authors: Singhal P. C. , Kapasi A. A. , Franki N. , Reddy K.
Published: March 2003

Laboratory and clinical reports indicate that opiate addicts are prone to infections. This effect of opiates is partly attributed to opiate‐induced macrophage (Mφ) apoptosis. In the present study, we evaluated the role of transforming growth factor‐β (TGF‐β) in morphine‐induced apoptosis of murine J774 cells and peritoneal Mφ. Mφ harvested from morphine‐treated mice showed greater (P < 0·0001) apoptosis when compared with control Mφ. Morphine also enhanced apoptosis of J774 cells and peritoneal Mφ. Anti‐TGF‐β antibody inhibited (P < 0·001) the morphine‐induced apoptosis in J774 cells (control 0·7 ± 0·4%; 10−6 m morphine 23·5 ± 0·7%; anti‐TGF‐β antibody (Ab) + 10−6 m morphine 8·1 ± 0·7%; apoptotic cells/field) and peritoneal Mφ (control 1·5 ± 0·9%; 10−6 m morphine 29·1 ± 1·4%; 10−6 m morphine + anti‐TGF‐β Ab 19·1 ± 1·8%; apoptotic cells/field). TGF‐β enhanced (P < 0·001) apoptosis of J774 cells and peritoneal Mφ. TGF‐β also promoted Mφ DNA fragmentation into integer multiples of 180 bp (ladder pattern). Immunocytochemical studies revealed that morphine enhanced the Mφ cytoplasmic content of TGF‐β. In addition, Western blotting showed increased production of TGF‐β by morphine‐treated J774 cells when compared with control cells. Morphine increased J774 cell expression of bax. Interestingly, morphine‐induced bax expression was inhibited by anti‐TGF‐β Ab. As both morphine‐induced J774 cell apoptosis and bax expression were inhibited by anti‐TGF‐β Ab, it appears that morphine‐induced J774 cell apoptosis may be mediated through the generation of TGF‐β.
Interleukin‐10 does not affect phagocytosis of particulate antigen by bonemarrow‐derived dendritic cells but does impair antigen presentation
Authors: Faulkner L. , Buchan G. , Baird M.
Published: March 2003

Dendritic cells (DC) are important initiators of an immune response so understanding the factors controlling antigen acquisition and presentation has important consequences for the use of these cells in vaccines and other forms of immunotherapy. We investigated the factors that influence phagocytosis by immature bone marrow‐derived DC (BMDC) and the effect of interleukin‐10 (IL‐10) on this process. Two sizes of fluorescent particles and recombinant bacillus Calmette–Guèrin expressing the green fluorescent protein (rBCG) were used as particulate antigens. The percentage of cells taking up the antigen was found to be dependent on the size and dose of the particles, and the length of exposure to them. BMDC exposed to IL‐10 at various concentrations for different periods exhibited no distinguishable change in antigen uptake. However, if BMDC treated with IL‐10 and rBCG were then exposed to a second dose of particulate antigen, uptake was increased compared with those BMDC not treated with IL‐10. The expression of major histocompatibility complex class II, CD80, CD86 and CD11c by BMDC after phagocytosing rBCG or inert beads, was inhibited when the BMDC were pretreated with IL‐10. In contrast, the expression of CD25 was increased. BMDC that had taken up BCG or purified protein derivative (PPD) were able to stimulate primed T‐cell proliferation but this was severely inhibited if the BMDC were cultured with IL‐10 before exposure to the antigen. This work suggests that although IL‐10 does not affect the phagocytic capacity of BMDC, it does inhibit maturation of the cells and consequently, T‐cell activation.
Common antigenicity between Japanese cedar (Cryptomeria japonica) pollen and Japanese cypress (Chamaecyparis obtusa) pollen, I. H‐2 complex affects cross responsiveness to Cry j 1 and Cha o 1 at the T‐ and B‐cell level in mice
Authors: Kingetsu I. , Ohno N. , Hayashi N. , Sakaguchi M. , Inouye S. , Saito S.
Published: March 2003

Common antigenicity among two purified Japanese cedar pollen allergens (Cry j 1 and Cry j 2) and one Japanese cypress pollen allergen (Cha o 1) was explored at the T‐cell and B‐cell level in mice of different H‐2 haplotypes. Cry j 2 did not show any common antigenicity with Cry j 1 or Cha o 1. B10.S (H‐2S) mice immunized with Cry j 1 or Cha o 1 generated T cells and antibodies reactive to both antigens, indicating the common antigenicity of these antigens. C57BL/6 (H‐2b) mice were non‐responders to Cry j 1. BALB/c (H‐2d) mice immunized with Cry j 1 or Cha o 1 and C57BL/6 mice immunized with Cha o 1 generated T cells that were only reactive with the respective immunogen, but produced antibody reactive to both Cry j 1 and Cha o 1, indicating that Cry j 1 and Cha o 1 share their B‐cell epitope but not their T‐cell epitope. This finding may provide a clue for the clarification of the T‐cell and B‐cell epitopes of Cry j 1 and Cha o 1, even though the data are influenced by H‐2 complex restriction in mice. Considering that H‐2 complex restriction affects cross responsiveness to Cry j 1 and Cha o 1 at the T‐ and B‐cell level in mice, we assessed the possible situation in humans exposed sequentially to Japanese cedar pollen and Japanese cypress pollen.
Immunoglobulin E antibodies of atopic individuals exhibit a broad usage of VH‐gene families
Authors: Eibensteiner P. , Spitzauer S. , Steinberger P. , Kraft D. , Valenta R.
Published: March 2003

The term ‘atopy’ describes the genetically determined tendency to mount immunoglobulin E (IgE) antibody responses against per se harmless antigens (allergens). In this study we investigated the usage of VH families in the formation of IgE antibodies in 10 patients suffering from mucosal and/or skin manifestations of atopy. IgE antibody reactivities to exogenous allergen sources as well as to autoallergens were determined and, by immunoabsorption, it was demonstrated that allergen‐specific IgE accounted for most of the total serum IgE levels in these patients. Using primers with specificity for the VH1–6 gene families and a primer specific for the first constant region of human IgE, cDNAs coding for IgE heavy chain fragments were amplified using the reverse transcription–polymerase chain reaction (RT–PCR) from peripheral blood lymphocytes of the 10 atopic individuals. Hybridization of the heavy chain‐encoding cDNAs with an IgE‐specific internal oligonucleotide probe revealed a broad usage of all VH‐gene families in the atopic individuals. The spectrum of VH families used in a given atopic individual was neither associated with the type or severity of clinical symptoms nor with the number of allergens recognized. The fact that allergen‐specific IgE antibodies in atopic individuals originate from a broad variety of B cells thus reflects the activation of multiple B‐cell clones during allergen sensitization. This finding should be borne in mind if therapeutic strategies for Type I allergy are considered that aim at a clonal elimination of allergen‐specific B cells.
Hydrogen peroxide augments eosinophil adhesion via β2 integrin
Authors: Nagata M. , Yamamoto H. , Shibasaki M. , Sakamoto Y. , Matsuo H.
Published: March 2003

During eosinophil (EOS) accumulation at sites of allergic inflammation, an initial step is the binding of EOS to adhesion molecules expressed on vascular endothelial cells (EC). We have previously observed that adhesion of peripheral blood EOS to recombinant human vascular cell adhesion molecule‐1 (rh‐VCAM‐1) stimulates the respiratory burst of EOS. Although the biological consequence of this activation remains to be elucidated, reactive oxygen species such as hydrogen peroxide (H2O2) may modify the adhesive property of EOS. In the present study, we examined whether H2O2 modifies the adhesive property of EOS. EOS were isolated from the peripheral blood of healthy subjects. Adhesion of the EOS to paraformaldehyde‐fixed human umbilical vein EC (HUVEC), stimulated or not stimulated with tumour necrosis factor‐α (TNF‐α; 100 p
m for 24 hr), was examined in the presence or absence of H2O2. H2O2 significantly enhanced adhesion of EOS to both resting and TNF‐α‐stimulated fixed HUVEC (P < 0·01, respectively). Such enhancing effects were inhibited by anti‐β2 integrin antibody or anti‐CD11b antibody, but not by anti‐CD11a or anti‐α4 integrin antibody. H2O2 also enhanced EOS adhesion to rh‐intracellular cell adhesion molecule‐1 (ICAM‐1) but not to rh‐VCAM‐1. Finally, H2O2 enhanced the expression of both CD11b and CD18 on EOS. These results indicate that H2O2 directly augments the adhesive property of EOS through β2 integrin.
Disaccharides generated from heparan sulphate or heparin modulate chemokine‐induced T‐cell adhesion to extracellular matrix
Authors: Hershkoviz R. , Schor H. , Ariel A. , Hecht I. , Cohen I. R. , Lider O. , Cahalon L.
Published: March 2003

We have found previously that disaccharides (DS) enzymatically generated from heparin or heparan sulphate can modulate tumour necrosis factor‐α (TNF‐α) secretion from immune cells in vitro and cell‐mediated immune reactions in vivo. Here, we show that such DS can modulate the adhesion and migration of human T cells. We found that certain heparin‐ and heparan sulphate‐derived DS induced, in a dose‐dependent manner, the adhesion of human T cells to both extracellular matrix (ECM) and immobilized fibronectin (FN); maximal T‐cell adhesion occurred with 1 ng/ml of DS. The levels of T‐cell adhesion to ECM that were induced by the tested DS molecules resembled those induced by the prototypic chemokine, macrophage inflammatory protein 1β (MIP‐1β). However, the kinetics of DS‐induced T‐cell adhesion to FN resembled that induced by phorbol myristate acetate (PMA), but not that induced by MIP‐1β. This adhesion appeared to involve β1 integrin recognition and activation, and was associated with specific intracellular activation pathways. Although a first exposure of T cells to certain DS molecules appeared to result in cell adhesion, a subsequent exposure of T cells to pro‐adhesive chemokines, such as MIP‐1β or RANTES, but not to other pro‐adhesive stimuli, for example interleukin‐2 or CD3 cross‐linking, resulted in inhibition of T‐cell adhesion to and chemotactic migration through FN. Hence, we propose that the breakdown products of tissues generated by inflammatory enzymes are part of an intrinsic functional programme, and not necessarily molecular waste. Moreover, because the DS molecules exert their modulatory functions within a limited time, it appears that the historical encounters of the tissue‐invading cells with the constituents of inflamed loci may dictate the cells’ behaviour upon subsequent exposure to proinflammatory mediators.
Production and functional characterization of a soluble recombinant form ofmouse CD59
Authors: Rushmere N. K. , Van Den Berg C. W. , Morgan B. P.
Published: March 2003

This report describes the engineering, expression, purification and functional characterization of a soluble recombinant form of murine CD59 (srMoCD59). We report the expression in Chinese hamster ovary (CHO) cells of a modified mouse CD59 cDNA that had been truncated at D‐74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six additional C‐terminal histidines. The expressed srMoCD59 was purified from tissue culture supernatant by means of its poly‐histidine tag using immobilized metal affinity chromatography. In comparison with CD59 on mouse erythrocytes, the srMoCD59 had a reduced molecular weight (18–20 000 as compared with 20–28 000 for GPI‐anchored srMoCD59). The terminal complement inhibitory capacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)‐triggered ‘reactive‐lysis’ system and a C5b‐7 site assay. In both assays, srMoCD59 inhibited lysis by the sera from all three species tested in the rank order mouse > rat >> human. The amount of srMoCD59 required to produce 50% inhibition of lysis in the C5b‐7 site assay, using purified terminal components to develop lysis, was 10‐fold less than that required in the same assay when EDTA serum was used as a source of C8 and C9, or in the CVF reactive lysis system. These data indicate that the presence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues as therapeutic agents in complement‐mediated diseases.