bluuebunny/crossref_metadata_2025 huggingface dataset:
The involvement of nitric oxide in the anti‐Candida albicans activity of rat neutrophils
Authors: FIERRO I. M. , BARJA‐FIDALGO C. , CUNHA F. Q. , FERREIRA S. H.
Published: November 2003

Rat peritoneal neutrophils (PMN) spontaneously release nitric oxide (NO) when incubated in vitro. Addition of the NO synthase inhibitor L‐monomethylarginine (L‐NMMA) to the PMN reduces NO production and impairs the killing of the yeast Candida albicans, both effects being reversed by L‐arginine. These data strongly suggest that oxidative metabolism of L‐arginine by PMN is involved in the candidacidal activity of these cells. Rat blood PMN, which do not produce significant amounts of NO, exhibit a reduced killing capacity compared with peritoneal cells, except when they are obtained from lipopolysaccharide (LPS)‐treated rats. In this case they produce measurable amounts of nitrite and express high fungicidal activity in vitro. Confirming the candidacidal activity of NO, the exposure of the C. albicans cultures to different concentrations of NO donors leads to a reduction in their survival. The candidacidal activity related to the NO pathway in rat PMN is phagocytosis dependent, since the activity can be inhibited by cytochalasin B. However, the oxidative products of oxygen released by rat PMN do not seem to be involved in their candidacidal activity, as incubation of the cells with phorbol myristate acetate (PMA) increases release of superoxide anion but does not affect the pattern of killing. Our results suggest that NO could be an important candidacidal pathway in rat neutrophils.
Covalent binding of C3b to tetanus toxin: influence on uptake/internalization of antigen by antigen‐specific and non‐specific B cells
Authors: VILLIERS M.‐B. , VILLIERS C. L. , JACQUIER‐SARLIN M. R. , GABERT F. M. , JOURNET A. M. , COLOMB M. G.
Published: November 2003

Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell‐mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen‐presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT‐C3b). The antigen‐presenting cells were TT‐specific (4.2) or non‐specific (BL15) Epstein–Barr virus (EBV)‐transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen‐specific and antigen non‐specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non‐specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross‐linking between membrane immunoglobulins and complement receptors on antigen‐specific cells. The consequences of covalent C3b binding to TT were analysed using antigen‐specific and antigen‐non‐specific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of non‐specific cells, similar to that observed in antigen‐specific cells. Thus, C3b covalently linked to antigen enlarges the array of B‐cell types capable of presenting antigen, including non‐specific cells.
Identification of macrophage migration inhibitory factor in murine neonatal calvariae and osteoblasts
Authors: ONODERA S. , SUZUKI K. , MATSUNO T. , KANEDA K. , KURIYAMA T. , NISHIHIRA J.
Published: November 2003

Bone resorption and formation are dynamic processes that occur in both normal and injured bone tissues. Regulation of these processes is mediated at the local level by cytokines and growth factors. Macrophage migration inhibitory factor (MIF) is one of the proinflammatory cytokines that activates macrophages and regulates production of other cytokines, such as tumour necrosis factor‐α and interleukin‐1. We here demonstrate, by reverse transcription–polymerase chain reaction, high expression of MIF mRNA in murine osteoblasts obtained from mouse neonatal calvariae and murine osteoblastic MC3T3‐E1 cells. The presence of MIF protein in the osteoblasts was confirmed by Western blot analysis using anti‐rat MIF antibody. Moreover, the immunohistochemical study revealed that MIF was localized largely in the cytoplasm. The pathophysiological function of MIF remains undefined; however, the present results suggest that MIF takes part in the osseous metabolism as well as in immunological events.
The autoimmunogenic chemicals HgCl2 and diphenylhydantoin stimulate IgG production to TNP–Ficoll and TNP–OVA, supporting and extending the graft‐versus‐host hypothesis for chemical induction of autoimmunity
Authors: ALBERS R. , VAN DER PIJL A. , SEINEN W. , PIETERS R. , BLOKSMA N.
Published: November 2003

Bypass of T‐cell tolerance via non‐cognate graft‐versus‐host (GVH)‐like help from T‐helper (Th) cells activated by chemically altered or induced epitopes, has been postulated as a mechanism underlying chemical induction of autoimmunity. To functionally test this hypothesis, we assessed whether the autoimmunogenic chemicals HgCl2 and diphenylhydantoin (DPH), like GVH reactions, stimulate specific immunoglobulin G (IgG) responses to trinitrophenyl (TNP)–Ficoll but not to TNP–ovalbumin. IgG responses were quantified in the popliteal lymph node by enzyme‐linked immunosorbent spot‐forming cell assay (ELISPOT) assays 7 days after s.c. injection of antigens, parental cells, chemicals or combinations thereof into the footpad of semi‐allogeneic F1 mice. Antigens, chemicals, or cells alone induced few TNP‐specific IgG antibody‐forming cell (AFC) compared with untreated mice. Co‐injection of parental cells or chemicals with TNP–Ficoll stimulated the TNP‐specific response per lymph node ∼50‐ and ∼40‐fold, respectively. In contrast, the IgG response to TNP–ovalbumin could not be stimulated by GVH reactions, whereas HgCl2 and DPH dose‐dependently increased this response up to ∼25‐ and ∼250‐fold, respectively. However, responses to TNP–ovalbumin pre‐incubated with HgCl2 or DPH could be stimulated ∼6–8 fold by GVH reactions. Observed similar adjuvanticity of chemicals and parental cells for TNP–Ficoll support a GVH‐like action of autoimmunogenic chemicals. In addition, the chemicals modify TNP–ovalbumin such that B cells recognizing this antigen become susceptible to non‐cognate stimulation by GVH reactions.
Neutrophils are involved in the non‐specific resistance to listeriosis induced by mycobacterial infections
Authors: LEAL I. S. , APPELBERG R. , SILVA M. T.
Published: November 2003

A major role has been recently ascribed to the neutrophil in the resistance to infection by Listeria monocytogenes (L. monocytogenes). Here we evaluated whether such neutrophils played a role in the non‐specific resistance to listeriosis that develops in hosts infected by mycobacteria. We found that the depletion of neutrophils completely abrogated the resistance conferred by the activated macrophages induced during the mycobacterial infection. The lack of killing by activated Kupffer cells and the visualization of bacteria proliferating inside peritoneal macrophages in neutrophil‐depleted mice allowed us to postulate a role for the cooperation between neutrophils and macrophages in the killing of L. monocytogenes. We also found listerial proliferation in hepatocytes of neutrophil‐depleted, mycobacteria‐infected mice showing that the neutrophils may be involved in the control of listeria infection of parenchymal cells.
The role of small intestinal antigen‐presenting cells in the induction of T‐cell reactivity to soluble protein antigens: association between aberrant presentation in the lamina propria and oral tolerance
Authors: HARPER H. M. , COCHRANE L. , WILLIAMS N. A.
Published: November 2003

The oral administration of soluble protein antigen results in profound immunological tolerance. However, the tissue location and function of antigen‐presenting cells (APC) that stimulate this response remain unclear. We have hypothesized that the properties of cells presenting antigen to naive T cells within the gut are involved, and therefore gut APC should stimulate T‐cell responses with different characteristics to those induced by other APC. To test this, we studied in vitro primary T‐cell responses following presentation of soluble protein antigen by cells from the Peyer's patches (PPC) and lamina propria (LPC) of the murine small intestine and the spleen (SPLC). Each APC population stimulated antigen‐specific proliferative responses with similar anamnestic characteristics; however, analysis of the cytokines produced revealed marked differences. Whereas SPLC stimulated the balanced production of T‐helper type 1 (Th1) and Th2 cytokines, PPC induced a profile consistent with the provision of T‐cell help for IgA production. Interestingly, presentation of antigen by LPC stimulated high levels of interferon‐γ (IFN‐γ) and transforming growth factor‐β (TGF‐β) in the absence of other cytokines [interleukin‐2 (IL‐2), IL‐4, IL‐5]. Evidence from analysis of cell activation and division within the cultures suggested that this profile may result from the preferential activation of CD8+ T cells by LPC; however, the lack of conventional CD4+ T‐cell cytokines indicated a defect in the normal function of these cells. Adoptive transfer of antigen‐pulsed LPC to syngeneic animals abrogated the induction of delayed‐type hypersensitivity (DTH) responsiveness, which followed a subsequent conventional antigen challenge further suggesting a role for lamina propria APC in tolerance induction.
The response of γδ T cells to Plasmodium falciparum is dependent on activated CD4+ T cells and the recognition of MHC class I molecules
Authors: MORRIS JONES S. , GOODIER M. R. , LANGHORNE J.
Published: November 2003

Peripheral blood γδ T cells from non‐exposed individuals respond to antigens of the malaria parasite, Plasmodium falciparum,in vitro. This response, largely caused by T cells bearing the Vγ9+ chain of the T‐cell receptor, is stimulated by components of the parasite expressed on the schizont stage and released at schizont rupture. The response of Vγ9+ T cells to parasite components is inhibited by antibodies to major histocompatibility complex (MHC) class I and class II. However, the inhibition by anti‐MHC class II antibodies can be overcome by the addition of interleukin‐2 (IL‐2) to the cultures, suggesting that γδ T cells themselves do not recognize MHC class II molecules but require an MHC class II‐dependent response taking place in the culture. In contrast, the inhibition by anti‐class I antibodies cannot be reversed by addition of IL‐2. Since an accompanying CD4+ T‐cell response occurred in peripheral blood mononuclear cells cultured with P. falciparum antigens, it was considered that these cells provide the cytokines necessary for the subsequent activation and expansion of Vγ9+ T cells recognizing components of the parasite and MHC class I molecules. This was confirmed by reconstituting the response of enriched γδ T cells to P. falciparum schizont extract by addition of purified CD4+ T cells.
Comparison of the immunoglobulin heavy‐chain complementarity determining region‐3 structure among the DNA sequences and the μ‐ and γ‐transcripts in human B‐lineage cells
Authors: KIYOI H. , MORI H. , HORIBE K. , OHNO R. , NAOE T.
Published: November 2003

To study the recombinational significance of the (immunoglobulin heavy) IgH chain gene in human B‐cell development, we compared the complementarity determining region (CDR)‐3 sequences of the DNA and the μ‐transcripts from human normal pre‐B cells and mature B cells, and the γ‐transcripts from bone marrow cells. The CDR‐3 sequences were longer in the DNA than in the μ‐ and γ‐transcripts, and this was independent of whether or not the rearrangement was productive. The DLR family genes were less frequently used in the μ‐ and γ‐transcripts. When translated into amino acids, all CDR‐3 sequences from the μ‐ and γ‐transcripts were productive, although 26.2% of the DNA sequences had stop codons in the D element and/or frameshifts of the Jh gene segments. The CDR‐3 of the productive DNA sequences in pre‐B cells frequently (26.6%) contained at least three continuous hydrophobic amino acids, which were mainly coded by the DLR and DXP family genes at the third reading frame. However, such motifs were rare in the μ‐transcripts of pre‐B (7.7%) and mature B cells (3.9%), and in the γ‐transcripts of bone marrow cells (1.1%) as well as in the DNA of mature B cells (10.4%). These findings suggested that the length and/or hydrophobicity of the IgH CDR‐3 might play a role in the selection mechanisms of B‐cell development.
Interferon‐γ promotes the survival and FcεRI‐mediated histamine release in cultured human mast cells
Authors: YANAGIDA M. , FUKAMACHI H. , TAKEI M. , HAGIWARA T. , UZUMAKI H. , TOKIWA T. , SAITO H. , IIKURA Y. , NAKAHATA T.
Published: November 2003

We examined the effects of interferon‐γ (IFN‐γ) on 100% pure human mast cells generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin‐6 (IL‐6). When mast cells were suspended in serum‐free medium without any cytokine after the withdrawal of SCF and IL‐6, they died over a period of 5 days because of apoptosis. IFN‐γ in the cultures suppressed apoptosis and prolonged their survival in a dose‐dependent manner. This survival‐promoting effect of IFN‐γ was blocked by neutralizing antibodies to IFN‐γ or to IFN‐γ receptor (IFN‐γR). When mast cells were incubated with IFN‐γ in serum‐free medium for more than 4 hr during sensitization, immunoglobulin E (IgE)/anti‐IgE antibody‐induced histamine release was effectively enhanced. Polymerase chain reaction (PCR) amplification of the α‐chain of IFN‐γR (IFN‐γRα) yielded products of the correct size predicted from the sequence of the receptor. In addition, flow cytometry using anti‐IFN‐γR monoclonal antibodies (mAbs) indicated that these mast cells bear IFN‐γR on their surface. These findings suggested that IFN‐γ activates human mast cells via specific receptors in certain aspects of inflammatory reactions.
Th0‐like CD4+ T cells protect mice with murine retrovirus‐induced immunodeficiency syndrome (MAIDS) against co‐infection with Listeria monocytogenes
Authors: HIROMATSU K. , NISHIMURA H. , KIMURA K. , AOKI Y. , USAMI J. , KOBAYASHI N. , MAKINO M. , YOSHIKAI Y.
Published: November 2003

We examined the host defence mechanism against infection with Listeria monocytogenes, a facultative intracellular bacterium, in mice with murine acquired immunodeficiency syndrome (MAIDS) caused by LP‐BM5 murine leukaemia virus (MuLv) infection. Although LP‐BM5 MuLV infection in C57BL/6 mice leads to a stage of immunodeficiency characterized by severe compromise of cell‐mediated immunity, the mice with established MAIDS infected with LP‐BM5 8 weeks previously, showed resistance to an intraperitoneal infection with Listeria monocytogenes. These MAIDS mice also showed resistance to a lethal dose of secondary listerial challenge, while the delayed‐type hypersensitivity response to heat‐killed Listeria (HKL) was severely impaired in MAIDS mice. The resistance of MAIDS mice to listerial infection was mediated by CD4+αβ T cells but neither by γδ T cells nor natural killer (NK) cells. Interferon‐γ (IFN‐γ) and interleukin‐10 (IL‐10) were produced by CD4+ T cells from Listeria‐infected MAIDS mice in response to the in vitro stimulation with HKL, whereas IFN‐γ but not IL‐10 were produced by those from Listeria‐infected control mice. These results suggest that T‐helper 0 (Th0)‐like immune responses of CD4+ T cells occur and participate in host defence mechanisms against listerial infection in MAIDS mice.
The emergence of non‐cytolytic NK1.1+ T cells in the long‐term culture of murine tumour‐infiltrating lymphocytes: a possible role of transforming growth factor‐β
Authors: TAMADA K. , HARADA M. , ITO O. , TAKENOYAMA M. , MORI T. , MATSUZAKI G. , NOMOTO K.
Published: November 2003

The mechanism by which murine tumour‐infiltrating lymphocytes (TIL) decreased their anti‐tumour activity during an in vitro culture with interleukin‐2 (IL‐2) was investigated. A phenotypic analysis revealed that the TIL cultured for 7 days (TIL‐d7) were exclusively NK1.1− CD4− CD8+ CD3+ cells and that this population was replaced by natural killer (NK)1.1+ CD4− CD8− CD3+ cells by day 27 (TIL‐d27) during the culture of TIL. The TIL‐d7 cells showed a cytolytic activity against B16 melanoma, whereas the TIL‐d27 cells had lost this activity, suggesting that the decrease in the anti‐tumour effect of TIL during the culture with IL‐2 was due to their populational change. Analysis on the characteristics of the TIL‐d27 cells revealed that they expressed skewed T‐cell receptor (TCR) Vβ5 and increased mRNA expression of Vα14. In addition, they expressed transforming growth factor‐β (TGF‐β) mRNA. Interestingly, TGF‐β augmented the proliferation of TIL‐d27 cells under the presence of IL‐2, but suppressed that of TIL‐d7 cells. Moreover, the proliferation of TIL‐d27 cells was suppressed by anti‐TGF‐β monoclonal antibody. Collectively, these results suggest that, in contrast to its suppressive effect on anti‐tumour effector T cells, TGF‐β could be an autocrine growth factor for NK1.1+ T cells and thereby induce non‐cytolytic NK1.1+ T cells in the long‐term culture of TIL.
Contribution of CD4+ and CD8+ T lymphocyte subsets to the cytokine secretion patterns induced in mice during sensitization to contact and respiratory chemical allergens
Authors: DEARMAN R. J. , MOUSSAVI A. , KEMENY D. M. , KIMBER I.
Published: November 2003

Chemical allergens of different types, those that cause in humans allergic contact dermatitis or occupational asthma, induce in mice divergent immune responses characteristic, respectively, of T‐helper 1 (Th1)‐ and Th2‐type cell activation. Such responses are associated with the development of different cytokine secretion patterns by draining lymph node cells (LNC), such that contact allergens stimulate vigorous interferon‐γ (IFN‐γ) production, but little secretion of the Th2 cytokines interleukin‐4 and interleukin‐10 (IL‐4 and IL‐10), whereas the converse pattern is provoked by respiratory allergens. Using selective depletion with antibody and complement we have here examined the relative contribution of CD4+ and CD8+ T lymphocytes to the cytokine secretion patterns of draining LNC isolated from mice sensitized to chemical allergens. Mice received repeated topical applications of respiratory allergens, trimellitic anhydride (TMA) or diphenylmethane diisocyanate (MDI), or of contact allergens 2,4‐dinitrochlorobenzene (DNCB) or formaldehyde. Thirteen days following the initiation of exposure the production by draining LNC of IL‐10, IFN‐γ and mitogen (concanavalin A)‐inducible IL‐4 was measured by enzyme‐linked immunosorbent assay (ELISA) after various periods of culture. It was found that the high levels of IL‐4 and IL‐10 secretion stimulated by TMA or MDI, and the lower levels of these cytokines induced by DNCB or formaldehyde, were in all cases dependent upon the presence of CD4+ cells. In contrast, the comparatively high concentrations of IFN‐γ observed following exposure to contact allergens were found to be derived from CD4+ cells, and in the case of DNCB from CD8+ cells also. The low levels of IFN‐γ induced by treatment with TMA or MDI were associated largely or wholly with CD8+ cells. These data indicate that the type 2 cytokine responses induced to different extents by both contact and respiratory chemical allergens are almost exclusively a function of CD4+ cells, but that IFN‐γ is produced by either CD4+ and CD8+ cells in the case of contact allergens or largely by CD8+ cells in the case of chemical respiratory allergens.
TNF‐α‐mediated expression of membrane‐type matrix metalloproteinase in rheumatoid synovial fibroblasts
Authors: MIGITA K. , EGUCHI K. , KAWABE Y. , ICHINOSE Y. , TSUKADA T. , AOYAGI T. , NAKAMURA H. , NAGATAKI S.
Published: November 2003

Degradation of the extracellular matrix plays an important role in rheumatoid articular destruction. Rheumatoid synovial fibroblasts secrete a large amount of matrix‐degrading metalloproteinases (MMPs), which initiate tissue damage by proteolytic degradation of collagens and proteoglycans. Cytokines, such as interleukin‐1α, ‐1β or tumour necrosis factor (TNF)‐α, are potent inducers of MMPs in rheumatoid synovial fibroblasts. MMPs are synthesized and secreted as latent pro‐enzymes and their activation is achieved by proteolytic cleavage of the propeptide domain at the N‐terminus of the molecule. Thus, the interaction of the pro‐enzymes with specific activators determines the enzymatic activity in the extracellular space. In the present study, we identified a novel mechanism for the activation of pro‐MMP‐2, which can be achieved through the interaction of the inflammatory cytokine, TNF‐α, with synovial fibroblasts. Although MMP‐2 is constitutively secreted by synovial fibroblasts as a pro‐enzyme, stimulation of fibroblasts by TNF‐α‐induced secretion of MMP‐2 in an active form. In support of this result, TNF‐α stimulation‐induced membrane‐type matrix metalloproteinase (MT‐MMP), a newly identified MMP‐2‐specific activator, on synovial fibroblasts. Cycloheximide analysis demonstrated that protein synthesis may be required for TNF‐α‐mediated MT‐MMP expression on synovial fibroblasts. Our results suggest that TNF‐α induces MMP‐2 activation in part by up‐regulating MT‐MMP expression, thus representing a new mechanism for cytokine‐mediated articular destruction in rheumatoid arthritis (RA).
Prevention of superantigen‐induced down‐regulation of T‐cell mediated cytotoxic activity by IL‐2 in vivo
Authors: BELFRAGE H. , DOHLSTEN M. , HEDLUND G. , KALLAND T.
Published: November 2003

Administration of staphylococcal enterotoxin A (SEA) to mice induces profound activation, cytokine production and cytotoxic activity of both CD4+ and CD8+ T cells, but subsequently activated cells are deleted or become anergic. This study demonstrates that administration of interleukin‐2 (IL‐2) can prevent sea‐induced hyporesponsiveness in CD8+ cytotoxic T lymphocytes (CTL). Repeated injections with sea every fourth day resulted in severly reduced cytotoxic activity in the spleen, which correlated with a reduced number of sea‐responsive T‐cell receptor (TCR)‐Vβ11+CD8+ cells. Studies of purified TCR‐Vβ11+CD8+ cells showed that they possessed intact cytotoxic activity per cell compared with cells from mice given a single injection of SEA, indicating that deletion was the main mechanism for the reduced cytotoxic activity. Combined treatment with SEA and IL‐2 increased the number of cytotoxic cells in the spleen after each SEA injection and prevented the down‐regulation of cytotoxic activity. Increased cytotoxic activity could be related to increased number and proliferation of CD8+IL‐2Rα+ cells, suggesting that administration of IL‐2 maintained IL‐2 responsiveness among CD8+ cells. Studies of sorted TCR‐Vβ11+CD8+ cells demonstrated that combined treatment with SEA and IL‐2 also increased cytotoxic activity per cell compared with treatment with SEA alone. Taken together, IL‐2 administration in vivo augmented SEA‐induced expansion of T cells as well as the cytotoxic activity per CTL, and prevented SEA‐induced cell deletion.
Binding of human and rat CD59 to the terminal complement complexes
Authors: LEHTO T. , MORGAN B. P. , MERI S.
Published: November 2003

CD59‐antigen (protectin) is a widely distributed glycolipid‐anchored inhibitor of complement lysis. CD59 interacts with complement components C8 and C9 during assembly of the membrane attack complex (MAC). To evaluate species specificity of these interactions we have in the present study examined cross‐species binding of isolated human and rat CD59 to the terminal complement components C8 and C9. By using primarily soluble CD59 isolated from urine (CD59U) potentially non‐specific binding interactions of the phospholipid portion of the membrane forms of CD59 could be avoided. Sucrose density gradient ultracentrifugation analysis showed that human CD59U bound to both human and rat C8 in the SC5b–8 complexes. Similar binding occurred when rat CD59U was used. The degree of binding did not significantly differ between the heterologous and homologous CD59–C8 combinations. C9 from both species inhibited the binding of CD59 to soluble SC5b–8. In ligand blotting analysis human and rat CD59U bound to human and rat C8αγ‐subunit and C9. Binding of human and rat CD59U was stronger to human than rat C9. In plate binding assays the erythrocyte form of CD59 (CD59E) bound to both human and rat C8. Binding of CD59E to heterologous C9 was considerably weaker than to homologous C9. Our results imply that the reciprocal binding sites between C8 and CD59 and to a lesser degree between CD59 and C9 are conserved between human and rat. Interactions of CD59 with the terminal C components are thus species selective but not ‘homologously restricted’.
Inhibition of human complement by β‐glycyrrhetinic acid
Authors: KROES B. H. , BEUKELMAN C. J. , VAN DEN BERG A. J. J. , WOLBINK G. J. , VAN DIJK H. , LABADIE R. P.
Published: November 2003

Licorice, the root extract of Glycyrrhiza glabra L., is used as a medicine for various diseases. Anti‐inflammatory as well as anti‐allergic activities have been attributed to one of its main constituents, glycyrrhizin. These activities are mainly ascribed to the action of the aglycone, β‐glycyrrhetinic acid. β‐Glycyrrhetinic acid has a steroid‐like structure and is believed to have immunomodulatory properties. To determine whether interference with complement functions may contribute to the immunomodulatory activity of β‐glycyrrhetinic acid, its effects on the classical and alternative activation pathways of human complement were investigated. We found that β‐glycyrrhetinic acid is a potent inhibitor of the classical complement pathway (IC50=35 μm), whereas no inhibitory activity was observed towards the alternative pathway (IC50>2500 μm). The anticomplementary activity of β‐glycyrrhetinic acid was dependent on its conformation, since the α‐form was not active. It was also established that naturally occurring steroids, e.g. hydrocortisone and cortisone, did not inhibit human complement activity under similar conditions. Detailed mechanistic studies revealed that β‐glycyrrhetinic acid acts at the level of complement component C2.
The role of complement receptor type 1 (CR1, CD35) in determining the cellular distribution of opsonized immune complexes between whole blood cells: kinetic analysis of the buffering capacity of erythrocytes
Authors: NIELSEN C. H. , MATTHIESEN S. H. , LYNG I. , LESLIE R. G. Q.
Published: November 2003

Erythrocytes (E) express complement receptor, type 1 (CR1, CD35), by which they bind opsonized immune complexes (IC) in competition with leucocytes expressing higher numbers of CR1 as well as other complement‐ and Fc‐receptors. This may prevent inappropriate activation of phagocytic cells. We examined the distribution on whole blood cells of preformed tetanus toxoid (TT)/human anti‐TT IC, opsonized in situ in 80% autologous serum. Binding to E occurred rapidly and reflected the kinetics of C3‐fragment incorporation into the IC. Among eight donors, expressing 180–361 CR1 per E, >90% of the cell‐bound IC were associated with E from 1 to 5 min of incubation, decreasing to 12±13% after 40 min. Upon comparison of the IC‐binding to leucocytes in whole blood with that of isolated leucocytes we found that E, despite their extensive early complex uptake, only reduced the IC‐deposition on polymorphonuclear leucocytes (PMN) by 61±26% after 30 seconds of incubation and 47±14% after 5 min. During the subsequent 10 min, this buffering capacity of E was essentially abolished. E restricted the initial IC‐binding to B cells by 73±19%, but from 3 min of incubation the presence of E promoted, in a CR1‐dependent manner, a progressive uptake via CR2 by the B cells. CR1 was the dominant receptor in the early IC‐uptake by B cells as well as PMN and monocytes, since CR1‐blockade inhibited the initial IC‐uptake by these populations in a preparation of isolated leucocytes suspended in serum by ≥84% after 30 seconds of incubation. We conclude, that E exert a substantial buffering effect on the IC‐deposition on PMN, monocytes and B cells, while CR1 is the dominant receptor in the uptake by these cells. However, this effect is short‐lived and less than expected from the proportion of IC bound to E. Moreover, E are efficient processors of IC‐attached C3b/iC3b fragments to C3dg as indicated by a pronounced enhancement by E of IC‐uptake via CR2 on B cells. We propose that this mechanism may play a role in preventing phagocyte activation via CR3.
The tumour associated cell surface antigen A6H is costimulatory for human CD4+ but not CD8+ T cells
Authors: LABUDA T. , PARRA E. , HEDLUND G. , KALLAND T. , DOHLSTEN M.
Published: November 2003

The A6H monoclonal antibody (mAb) recognizes a 120 000–140 000 MW antigen that is expressed at similar densities on 85–90% of human CD4+ and CD8+ T cells and on renal cell carcinomas. The binding of the A6H mAb induced a costimulatory signal in anti‐CD3 activated T cells. In the present report, we show that A6H costimulated cell proliferation and cytokine production in purified CD4+ T cells. Unexpectedly, the CD8+ T‐cell subpopulation failed to respond. CD4+ T cells costimulated with the A6H mAb upregulated CD80, CD86, CD71, interleukin‐2 (IL‐2)Rα, IL‐2Rβ and IL‐2Rγ, while no corresponding up‐regulation of these cell surface molecules was seen in CD8+ T cells. In order to investigate the nature of the A6H mAb costimulus at the transcriptional level we have examined induction of the transcription factors OCT‐1, AP‐1 and NF‐κB which are known to be transcriptional regulators of several cytokine and cytokine receptor genes, including the IL‐2 and IL‐2R genes. Co‐ligation of the A6H antigen and the CD3 complex induced expression of the transcription factor AP‐1 in CD4+ T cells, whereas no increase in NF‐κB and octamer‐binding (Oct) proteins was seen compared to T cells stimulated with anti‐CD3 alone. Furthermore, no induction of AP‐1 was seen in A6H costimulated CD8+ T cells. These results suggests that both proximal steps in CD8+ T‐cell activation as well as the later phases are unresponsive to A6H ligation. Molecular differences of the A6H molecule or distinct regulation of the A6H transduced AP‐1 activation pathway may exist in CD4+ and CD8+ T cell subpopulations.
Selective migration of highly differentiated primed T cells, defined by low expression of CD45RB, across human umbilical vein endothelial cells: effects of viral infection on transmigration
Authors: BORTHWICK N. J. , AKBAR A. N. , MACCORMAC L. P. , LOWDELL M. , CRAIGEN J. L. , HASSAN I. , GRUNDY J. E. , SALMO M. , YONG K. L.
Published: November 2003

Low expression of CD45RB on CD45RO+ T lymphocytes defines a subset of highly differentiated T lymphocytes that accumulate in vivo within the affected joints of patients with rheumatoid arthritis (RA). Although it is known that CD45RO+ T lymphocytes migrate to sites of inflammation in vivo, it is not clear whether within this subset the CD45RBlo cells are selectively recruited or develop in situ within the joint. Using a transwell system we show that a small proportion of resting T lymphocytes migrated across unactivated human umbilical vein endothelial cells (HUVEC). These migrating cells were CD45RO+ and enriched for low CD45RB expression. In addition, both the CD45RO+CD45RBlo subset and migrating cells expressed increased levels of β1 and β2 integrins and CD44. The percentage of CD45RO+CD45RBlo T lymphocytes was increased in the circulation of patients with acute Epstein–Barr virus (EBV) infection. These in vivo activated cells also expressed increased levels β1 and β2 integrins and CD44, and showed an enhanced rate of transmigration compared with resting T lymphocytes. Transmigration of T lymphocytes was increased using the chemokines RANTES and lymphotactin and the cytokine interleukin‐15 (IL‐15). In addition, infection of the HUVEC with cytomegalovirus (CMV) led to an enhanced movement of T lymphocytes. In all of these cases the selective migration of the CD45RBlo subset was maintained. Thus although the rate of T‐lymphocyte transmigration could be influenced by a number factors, the CD45RO+CD45RBlo subset has a migratory advantage suggesting that more differentiated CD45RO+CD45RBlo T lymphocytes are selectively recruited to sites of inflammation.
Cloning, expression and biological function of the bovine CD40 homologue:‘qc role in B‐lymphocyte growth and differentiation in cattle‘pa
Authors: HIRANO A. , BROWN W. C. , ESTES D. M.
Published: November 2003

Members of the tumour necrosis factor receptor superfamily play a key role in B‐lymphocyte survival, proliferation, differentiation and programmed cell death. A member of this superfamily, the CD40 molecule, plays an important role in the differentiation of B lymphocytes into effector cells and in early activation through cognate interaction with T lymphocytes. In this report, we describe a cDNA and its protein product identified in cattle with approximately 70% sequence conservation at the nucleic acid level with the human CD40 gene. Transcripts for the boCD40 molecule were identified in resting and activated B lymphocytes, some but not all CD4+ and CD8+ T lymphocyte clones, and peripheral blood‐derived T lymphocytes. Coculture of resting B cells with simian virus 40 (SV40)‐transformed NIH3T3 (MOP8) cells stably transfected with the ligand for CD40 (bovine CD40 ligand, boCD40L), resulted in proliferation which was enhanced by addition of rbo interleukin‐4 (IL‐4). Cross‐linkage of CD40 on bovine B lymphocytes upon coculture with CD40L‐transfected cells resulted in the increased production of secretory IgM and, to a lesser extent, of IgG. Addition of rboIL‐4 to these cultures increased levels of IgM and IgG secretion approximately twofold over those induced by CD40L alone. Our results indicate that many of the functions described for human and mouse CD40 are also conserved in the bovine but that differences in subset distribution of expression of the CD40 molecule in lymphoid cell types in cattle may impact on regulation of the early activation steps in the acquired immune response.
Two distinct pathways of human macrophage differentiation are mediated by interferon‐γ and interleukin‐10
Authors: OLIKOWSKY T. , WANG Z.‐q. , DUDHANE A. , HOROWITZ H. , CONTI B. , HOFFMANN M. K.
Published: November 2003

Forming cellular conjugates with T cells, macrophages can help their targets to mount an immune response or they can destroy the targeted T cell. The two functions are performed by two distinct macrophage subsets that can be distinguished by cell surface marker phenotypes, B7+ CD16− and B7− CD16+. Interferon‐γ (IFN‐γ) induces the former, interleukin‐10 (IL‐10) induces the latter phenotype. The two macrophage differentiation pathways are mutually exclusive; each cytokine inhibits the effect of the other cytokine. The second messenger cAMP enhances the macrophage B7 expression and suppresses the macrophage CD16 expression. However, together with IL‐10, cAMP blocks the generation of both macrophage phenotypes. In the chimpanzee we noted deviations from this differentiation pattern that are suggestive of an enhanced IL‐10 presence in the primate environment.
Transcriptional activation of the interleukin‐8 gene by platelet‐activating factor in human peripheral blood monocytes
Authors: DENAULT S. , APRIL M. J. , STAŇKOVÁ J.
Published: November 2003

Interleukin‐8 (IL‐8) is a member of the chemokine family and a potent neutrophil chemoattractant and activator. It is produced by a variety of cell types during inflammation. In the present work, we examined the regulation of IL‐8 gene expression in monocytes by the pro‐inflammatory lipid mediator, platelet‐activating factor (PAF). Stimulation of human peripheral blood monocytes with PAF augmented their release of IL‐8. The enhancement of IL‐8 secretion was associated with an increase in IL‐8 mRNA expression. PAF induced a concentration‐ and time‐dependent augmentation of IL‐8 mRNA accumulation. The response was maximal at PAF concentrations of 10–100 nm. The increased mRNA expression was evident after 1·5 hr of stimulation and persisted for 6 hr. Stimulation of monocytes with PAF, followed by arrest of de novo transcription with actinomycin D, indicated that PAF only marginally increased the stability of IL‐8 mRNA. However, in vitro nuclear transcription demonstrated that the enhancement of IL‐8 mRNA expression occurred mainly at the transcriptional level. The PAF‐induced increase in IL‐8 mRNA levels could be blocked with a PAF receptor antagonist. These results show, for the first time, that IL‐8 gene expression and protein production can be upregulated by PAF. This interaction could be important in the development and amplification of the inflammatory response.
Generation of dendritic cell‐like antigen‐presenting cells in long‐term mixed leucocyte culture: phenotypic and functional studies
Authors: GAO J.‐X. , MADRENAS J. , ZENG W. , ZHONG R. , GRANT D.
Published: November 2003

The mechanisms contributing to the proliferation and differentiation of antigen‐presenting cell (APC) precursors upon antigen stimulation or tissue injury are poorly understood. Herein, we report the induction of a population of dendritic cell‐like cells (DLC) with potent antigen‐presentation function from unfractionated spleen cells by means of repetitive allostimulation in long‐term mixed leucocyte cultures (LT‐MLC). Initially, only a few adherent DLC were observed. By 4–6 weeks, however, there were large numbers of DLC which survived persistently. Features of these DLC are closely related to dendritic cells (DC), including: (1) dendritic, veiled or spiny‐processed morphology; (2) expression of a wide array of leucocyte surface markers including DC‐associated or restricted antigens: 33D1, NLDC‐145, CD11c (N418), heat‐stable antigen (HSA), CD44, B7‐1 and B7‐2; (3) ability to migrate to draining lymph nodes and white pulp area of spleen; (4) expression of high level of major histocompatability complex (MHC) class II molecules and (5) more potent mixed leucocyte reaction (MLR)‐stimulating capacity than peritoneal macrophages and APC‐enriched spleen cells. DLC‐stimulated MLR was inhibited by monoclonal antibodies (mAbs) to B7‐1, B7‐2, intracellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), leucocyte‐function associated antigen‐1 (LFA‐1) or very‐late activation antigen‐4 (VLA‐4) by 30–55%. When maintained for more than 2 months, the DLC did not lose their MLR‐stimulating activity, but many surface markers were down‐regulated except for Mac‐2 and VCAM‐1, which remained stable or were up‐regulated, respectively. In short‐term culture, the addition of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) or interleukin (IL)‐2 enhanced proliferation of DLC, while tumour necrosis factor‐α (TNF‐α) and IL‐4 did not. IL‐4 suppressed not only ‘spontaneous’, but also GM‐CSF‐enhanced proliferation, suggesting that cytokines play a differential role in DLC proliferation. These results confirm that professional APC can proliferate in response to repetitive antigen stimulation, and their proliferation is differentially regulated by cytokines. A comparison study of DLC with typical DC is being carried out in our laboratory.
TCR γδ+ cells are prominent in normal bovine skin and express a diverse repertoire of antigen receptors
Authors: HEIN W. R. , DUDLER L.
Published: November 2003

More than 80% of T cells in bovine skin localized in the superficial 0·5 mm of the dermis. Only 3% occurred within the epidermis or made contact with the stratum basale while the remainder occupied deeper dermal sites. The γδ T‐cell receptor (TCR) was expressed by 44% of T cells in skin and 39% and 35% expressed, respectively, the CD4 and CD8 markers. Some cells co‐expressed CD8 and the γδ TCR. A highly diverse repertoire of γδ TCR was expressed in skin due mainly to the usage of multiple Vδ segments and to extensive sequence variation at the junctions of both TCRγ and TCRδ chains. However, a single receptor isotype was used. Transcripts encoding several new components of the bovine γδ TCR were identified, including three new Vγ segments, the Cγ5 region and 13 new functional Vδ segments. Taken together with earlier findings, these results emphasize that ruminant γδ T cells express exceptionally diverse antigen receptors and suggest they may have a more elaborate recognitive capacity than do their counterparts in other species.
Reconstitution of SCID mice with haemopoietic precursors: a detailed analysis of γδ T‐cell reconstitution
Authors: KELLY K. A. , O’BRIEN R. , BORN W.
Published: November 2003

A well‐known characteristic of γδ T cells is that they are produced in waves during ontogeny, with cells expressing T‐cell receptor Vγ5 appearing early in fetal thymic ontogeny, followed by Vγ6, then by other γδ T‐cell types. In addition, evidence exists to suggest that the potential of haemopoietic precursors to generate different types of γδ T cells changes in ontogeny. We have used these observations as the basis for an extensive study of the potential for haemopoietic precursors isolated from fetal liver, neonatal spleen and adult bone marrow to reconstitute severe combined immunodeficient (SCID) mice. Mice that were reconstituted as newborns with fetal liver cells most closely resembled normal C.B‐17 mice with respect to both lymphocyte numbers and subsets, while mice reconstituted with adult bone marrow had fewer cells than normal mice. This deficit spanned both T and B cells in all organs examined. Among the γδ T‐cell subsets examined, the ability to reconstitute Vγ4+ cells was particularly dependent on the ontogenic age of the reconstituting presursors, with fetal liver cells having the greatest capacity to generate Vγ4+ cells, and adult bone marrow cells the least. The vast majority of the T cells produced in the reconstituted mice were of donor origin, and the level of reconstitution was found to be dependent upon some factor other than the presursor frequency.
Cytotoxic T‐lymphocyte interaction with fibronectin and vitronectin: activated adhesion and cosignalling
Authors: YBARRONDO B. , O’ROURKE A. M. , MCCARTHY J. B. , MESCHER M. F.
Published: November 2003

Stimulation of cloned cytotoxic T lymphocytes (CTL) with anti‐T‐cell receptor (TCR) monoclonal antibody (mAb) in solution resulted in rapid and sustained activation of adhesion to immobilized fibronectin (FN) but did not initiate degranulation. Addition of a second antibody (Ab) to further cross‐link the TCR substantially increased the level of adhesion and also activated degranulation, as measured by release of serine esterase, in the presence of immobilized FN but not in its absence. Thus, binding to FN can provide a costimulatory signal to activate degranulation. TCR cross‐linking also activated CD8‐dependent adhesion to class I, and CD8 provided a costimulatory signal upon binding to class I. However, the requirements for activating adhesion and generating the costimulatory signal differed significantly for FN versus class I ligand, suggesting that these two receptor–ligand systems do not share a common mechanism of action. Co‐immobilizing FN and alloantigen resulted in increased serine esterase release in comparison with that stimulated by antigen alone, and required the FN and class I be on the same surface. Peptide and antibody blocking demonstrated that CTL binding to FN, and to vitronectin (VN), was mediated by the αVβ3 vitronectin receptor (VNR). Thus, VNR is activated by a signal from the TCR to mediate adhesion to FN or VN, and delivers a costimulatory signal for degranulation via a different mechanism than costimulation by CD8 binding to class I.
Infection of C3HeB/FeJ mice with the docile strain of lymphocytic choriomeningitis virus induces autoantibodies specific for erythrocyte Band 3
Authors: MAZZA G. , EL AZAMI EL IDRISSI M. , COUTELIER J. P. , CORATO A. , ELSON C. J. , PFAU C. J. , DAY M. J.
Published: November 2003

C3HeB/FeJ mice infected with the docile strain of lymphocytic choriomeningitis virus (LCMV‐d) develop a persistent infection with a transient haemolytic anaemia. Immunoglobulin can be eluted from the red blood cells (RBC) of these mice but it cannot be detected on the RBC by a conventional antiglobulin test. The present study demonstrates that RBC from such mice bear erythrocyte autoantibodies which are predominantly of the IgG2a subclass, with lower levels of autoantibodies of the IgG1, IgG2b and IgG3 subclasses. To identify the target antigen the autoantibodies were eluted from the RBC of LCMV‐infected mice. The eluted autoantibody bound to intact normal RBC and precipitated a 105 000 MW component that corresponds to murine Band 3 protein. A monoclonal antibody derived from mice infected with LCMV‐d also precipitated mouse Band 3, and reacted specifically by enzyme‐linked immunosorbent assay against a purified preparation of Band 3. This study has shown that in C3H mice infected with LCMV‐d which develop autoimmune haemolytic anaemia, the target autoantigen is erythrocyte membrane Band 3.
The involvement of macrophage‐derived tumour necrosis factor and lipoxygenase products on the neutrophil recruitment induced by Clostridium difficile toxin B
Authors: SOUZA M. H. L. P. , MELO‐FILHO A. A. , ROCHA M. F. G. , LYERLY D. M. , CUNHA F. Q. , LIMA A. A. M. , RIBEIRO R. A.
Published: November 2003

Clostridium difficile (Cd) toxins appear to mediate the inflammatory response in pseudomembranous colitis and/or colitis associated with the use of antibiotics. In contrast to Cd Toxin A (TxA), Cd Toxin B (TxB) has been reported not to promote fluid secretion or morphological damage in rabbits and hamsters and also does not induce neutrophil chemotaxis in vitro. However, TxB is about 1000 times more potent than TxA in stimulating the release of tumour necrosis factor‐α (TNF‐α) by cultured monocytes. In the present study, we investigated the ability of TxB to promote neutrophil migration into peritoneal cavities and subcutaneous air‐pouches of rats. We also examined the role of resident peritoneal cells in this process as well as the inflammatory mediators involved. TxB caused a significant and dose‐dependent neutrophil influx with a maximal response at 0·1 μg/cavity after 4 hr. Depleting the peritoneal resident cell population by washing the peritoneal cavity or increasing this population by pretreating the animals with thioglycollate blocked and amplified the TxB‐induced neutrophil migration, respectively. Pretreating the animals with MK886 (a lipoxygenase inhibitor), NDGA (a dual cyclo‐ and lipoxygenase inhibitor) or the glucocorticoid, dexamethasone, but not with indomethacin (a cyclo‐oxygenase inhibitor), or BN52021 (a platelet‐activating factor antagonist), inhibited the neutrophil migration evoked by TxB. Pretreatment with dexamethasone or the administration of anti‐TNF‐α serum into the air‐pouches also significantly reduced the TxB‐induced neutrophil migration. Supernatants from TxB‐stimulated macrophages induced neutrophil migration when injected into the rat peritoneal cavity. This effect was attenuated by the addition of either MK886 or dexamethasone to the macrophage monolayer and by preincubating the supernatants with anti‐TNF‐α serum. TxB also stimulated the release of TNF‐α by macrophages. Overall, these results suggest that TxB induces an intense neutrophil migration which is mediated by macrophage‐derived TNF‐α and lipoxygenase products.