bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of human interleukin‐12 production by pentoxifylline
Authors: MOLLER D. R. , WYSOCKA M. , GREENLEE B. M. , MA X. , WAHL L. , TRINCHIERI G. , KARP C. L.
Published: November 2003

Pharmacological control of interleukin‐12 (IL‐12) production may be a key therapeutic strategy for modulating immunological diseases dominated by type‐1 cytokine responses. In this study, we investigated the effects of pentoxifylline on the production of IL‐12 by human blood mononuclear cells and primary human monocytes stimulated with heat‐killed Staphylococcus aureus Cowan strain I (SAC) or lipopolysaccharide (LPS). Pentoxifylline potently suppressed production of IL‐12 in a concentration‐dependent manner. In these same experiments, tumour necrosis factor‐α (TNF‐α) production was inhibited and IL‐10 and prostaglandin E2 (PGE2) production was enhanced by treatment with pentoxifylline. Suppression of IL‐12 production by pentoxifylline was found to be independent of several known endogenous inhibitors of IL‐12, such as IL‐10, transforming growth factor‐β (TGF‐β), IL‐4 and PGE2. RNase protection assays revealed that pentoxifylline inhibited accumulation of both IL‐12 p40 and p35 mRNA, suggesting a predominant mRNA locus for pentoxifylline‐induced IL‐12 inhibition. Low levels of pentoxifylline added to the suppression of IL‐12 production by suboptimal inhibiting doses of dexamethasone, suggesting that this drug combination may have therapeutic utility. These results provide a firm rationale for the use of pentoxifylline in clinical trials of immunological disorders characterized by inappropriate type‐1 immune responses.
Heterogeneity of cytokine functions in HIV infection
Authors: KUNDU S. K. , MERIGAN T. C.
Published: November 2003

Immunological responses, especially cytokines, play important roles in determining the persistence of infectious agents in chronic diseases. Th1 responses enhance cellular immunity to control infection whereas Th2 immune responses down‐regulate these effector immune responses. It has been suggested that the Th1 to Th2 switch is involved in human immunodeficiency virus (HIV) disease progression. We studied the regulatory role of interleukin‐4 (IL‐4; Th2 response) on interferon‐γ (IFN‐γ; Th1 response) in HIV infection and its role in the generation of HIV‐specific cytotoxic T lymphocytes (CTL) in an in vitro system. Forty HIV‐infected, asymptomatic individuals and 20 HIV‐seronegative individuals were included in this study. Peripheral blood mononuclear cells were stimulated with phytohaemagglutinin and tetanus toxoid in the presence or absence of IL‐4 to determine the effect of IL‐4 on IFN‐γ production and HIV‐Env‐specific CTL activity. IL‐4 showed a dual effect on IFN‐γ production in HIV patients. IL‐4 down‐regulated IFN‐γ production in HIV‐seronegative individuals and in 55% of HIV patients whereas it stimulated IFN‐γ production in 45% of HIV patients. IL‐4 increased HIV‐Env‐specific CTL activity in five of seven patients of the latter group. IL‐4 has multiple biological activities, e.g. IL‐4 inhibits IFN‐γ production as well as stimulates CTL generation which in turn produces IFN‐γ. Understanding the biological significance of these interactions is of importance for immunotherapeutic approaches against HIV infection.
Immune enhancing effects of dehydroepiandrosterone and dehydroepiandrosterone sulphate and the role of steroid sulphatase
Authors: SUITTERS A. J. , SHAW S. , WALES M. R. , PORTER J. R. , LEONARD J. , WOODGER R. , BRAND H. , BODMER M. , FOULKES R.
Published: November 2003

Steroid hormones, such as glucocorticoids (GC), influence immune and inflammatory responses through their suppressive actions. Recent evidence suggests that another steroid hormone, dehydroepiandrosterone (DHEA), provides an immunostimulatory influence opposing the effect of GC. DHEA circulates in its inactive sulphated form, DHEAS, requiring conversion to DHEA by a steroid sulphatase (SS) enzyme for biological activity. Therefore, inhibition of SS activity may affect immune responses, allowing endogenous GC effects to predominate. We have shown that administration of DHEA and DHEAS in contact sensitization (CS) augments ear swelling by 39 and 46% respectively (P<0·001). DHEAS at doses of 0·5, 5 and 50 mg/kg reverses the inhibitory effect of corticosterone (5 mg/kg) (P<0·01). In CS, CT2251 (SS inhibitor) at 10 and 0·1 mg/kg inhibited ear swelling by 61 and 38% (P<0·05) respectively. In addition, it inhibited DHEAS‐augmented responses by 49 and 35% respectively (P<0·05), with no effect on DHEA‐augmented responses. DHEAS reversed CT2251 inhibition of the CS response with complete reversal at 50 mg/kg (P<0·05). DHEAS and CT2251 appear to affect cellular infiltration into the ear, since DHEAS increased the number of lymphocytes by 63·8% and macrophages by 107% (P<0·001), whereas CT2251 at 0·1 mg/kg decreased the number of lymphocytes by 65% (P<0·001) and macrophages by 80% (P<0·001). DHEAS, CT2251 and dexamethasone had no effect on oedema in the ear. From our data we have shown that steroid hormones, such as DHEA, have the potential to act as immunostimulatory factors in vivo. Inhibiting the conversion of DHEAS to DHEA by SS enzyme leads to an anti‐inflammatory effect.
Epitope specificity of IgE antibodies to a major allergen (Cry j 1) of Japanese cedar pollen in sera of humans and monkeys with pollinosis
Authors: SAKAGUCHI M. , HASHIMOTO M. , NIGI H. , YASUEDA H. , TAKAHASHI Y. , WATANABE M. , NAGOYA T. , TANIGUCHI Y. , KURIMOTO M. , INOUYE S.
Published: November 2003

Japanese cedar (Cryptomeria japonica) pollinosis has been reported to occur naturally in Japanese monkeys (Macaca fuscata) as well as humans. Using monoclonal antibodies (mAb) specific to Cry j 1, a major allergen in Japanese cedar pollen, we identified five independent epitopes (EP‐1 to EP‐5) on the molecule. The epitopes recognized by IgE antibodies in the sera of humans and monkeys with the pollinosis were analysed by an IgE enzyme‐linked immunosorbent assay inhibition method with these mAb. In human patients, the mAb to EP‐1 strongly blocked the binding of IgE antibodies in all patients’ sera to Cry j 1. The reaction patterns of IgE antibodies in monkeys, however, varied among the troops of monkeys. In some troops, the mAb to EP‐1 showed a blocking pattern similar to that for human patients. In other troops, mAb to EP‐4 and EP‐5 blocked binding of IgE. These results indicate that some, but not all, monkeys have antibody responses to the major allergen similar to those of humans.
Differential regulation of IFN‐γ, IL‐10 and inducible nitric oxide synthase in human T cells by cyclic AMP‐dependent signal transduction pathway
Authors: BENBERNOU N. , ESNAULT S. , SHIN H. C. K. , FEKKAR H. , GUENOUNOU M.
Published: November 2003

Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signalling pathways. In the present work, we attempted to clarify the role of cAMP on interferon‐γ (IFN‐γ), interleukin (IL)‐10, IL‐4 and IL‐13 expression as well as on the inducible nitric oxide synthase (iNOS) expression. Treatment of phytohaemagglutinin (PHA)/phorbol 12‐myristate 13‐acetate (PMA)‐activated Jurkat cells with either dibutyryl‐cyclic adenosine monophosphate (cAMP) or pentoxifylline induced a strong inhibition of IFN‐γ mRNA expression as measured by reverse transcription (RT)–polymerase chain reaction (PCR), without affecting IL‐10 expression. Both cholera toxin and prostaglandin E2 (PGE2) induced a strong inhibition of IFN‐γ mRNA expression, whereas IL‐10 mRNA expression was significantly enhanced. This differential regulation of IFN‐γ and IL‐10 expression was related to intracellular cAMP concentration. IL‐13 and IL‐4 mRNA expressions were not inhibited. We developed a new method based on immunofluorescence for intracellular cytokine detection followed by optical and computerized image processing, and our results showed that IFN‐γ protein was strongly inhibited when cells were treated with PGE2 or dibutyryl (db)‐cAMP, whereas IL‐10 protein was enhanced. This suggests that cAMP exerts its action at both the transcriptional and protein levels. iNOS mRNA expression was markedly elevated in the presence of PGE2. The generation of nitric oxide using sodium nitroprusside (SNP) induced a dramatic decrease of IFN‐γ, while IL‐10 was enhanced; and conversely the inhibition of iNOS activity using l‐NG‐monomethyl arginine (l‐NMMA) induced a clear inhibition of IL‐10 and IL‐4, while IFN‐γ was enhanced. These results provide evidence that the protein kinase A (PKA) activation pathway plays a prominent role in the balance between the type 1 and type 2 cytokine profile in PHA/PMA‐activated Jurkat cells. Data also suggest that iNOS expression is under the control of PKA activation, and that NO seems to be able to assume the polarization of activated T cells to the type 2 profile.
Selection of antibodies to cell surface determinants on mouse thymic epithelial cells using a phage display library
Authors: PALMER D. B. , GEORGE A. J. T. , RITTER M. A.
Published: November 2003

The network of thymic epithelium contributes significantly to the thymic stromal cell environment, which plays a vital role in the generation and maturation of thymocytes. Monoclonal antibodies (mAb) have revealed considerable heterogeneity within this epithelial component of the mouse thymic microenvironment, but many of these antibodies recognize epitopes that are located inside the cell and so cannot be used in functional studies. As an alternative approach to isolate antibodies specific to thymic epithelium, we used a phage display library expressing single chain Fv antibodies. For selection, a thymic cell suspension was incubated with the phage display library, and major histocompatibility complex class II positive cells, the majority of which are epithelial, were then specifically selected. Phage bound to these cells were eluted and the selection procedure was repeated for a further five rounds. Immunohistochemical analysis revealed that these phage antibodies show differential staining of thymic epithelial subsets. Flow cytometric analysis of a thymic epithelial cell line using a panel of these antibodies demonstrated that they recognize epitopes on the cell surface. Furthermore, some of these antibodies also labelled human thymic epithelium, suggesting that the epitopes recognized by these antibodies are conserved between human and rodent thymus. Our approach therefore provides a rapid method to select antibodies specific for thymic epithelial cell surface determinants in their native configuration.
Antigen dose‐dependent predominance of either direct or sequential switch‘qc in IgE antibody responses
Authors: SUDOWE S. , RADEMAEKERS A. , KÖLSCH E.
Published: November 2003

Priming of CBA/J mice with minute doses of protein antigens (Ag) leads to high IgE antibody (Ab) titres in the immune sera of these animals. In contrast priming with large doses elicits only a marginal production of IgE Ab. In vitro restimulation of spleen cells from animals primed with large doses and lacking in vivo IgE Ab leads to a burst of IgE Ab‐forming cells. This in vitro anamnestic response is lacking in mice primed with minute doses of Ag. In order to trace the cellular basis of the in vitro IgE memory response we have extended the analysis of the distribution of Ab isotypes to Ag‐primed IgG1‐deficient Δ5′Sγ1 mice. The data presented here must be interpreted as followed. Priming of mice with minute doses of Ag leads to a direct switch from IgM to IgE Ab expression in both strains. These animals have high IgE Ab titres without establishing an IgE memory. The direct switch was verified by polymerase chain reaction and Southern blot analysis of switch circle DNA isolated from Ag‐specific B cells of CBA/J mice primed with minute doses of Ag. In contrast to immunization with minute doses, priming with large doses of Ag fails to induce in vivo IgE Ab production in CBA/J and Δ5′Sγ1 mice but establishes a Bɛ memory in CBA/J mice which involves IgG1‐bearing intermediate B cells. In vivo these Bɛ memory cells do not enter the status of IgE Ab‐producing cells. In vitro they can be released from this anergy and presumed suppression and develop in an anamnestic response into a large population of IgE Ab‐forming B cells. This increase in the number of IgE Ab‐producing cells after restimulation in vitro is lacking in Δ5′Sγ1 mice, apparently because of their inability to generate IgG1‐expressing precursor cells. The notion of a sequential switch and an IgG1 intermediate Bɛ memory status is also supported by depletion and inhibition experiments. Elimination of IgG1‐expressing B cells in CBA/J mice primed with high doses of Ag prevents the IgE Ab burst after in vitro challenge with Ag. The data further suggest that the two switch pathways are not mutually exclusive and that the Ag dose can decide which pathway is preferentially used.
Recipient age determines the success of intraperitoneal transplantation of peritoneal cavity B cells
Authors: JULIUS Jr* P. , KAGA† M. , PALMER‡ Y. , VYAS§ V. , PRIOR‡ L. , DELICE D. , RIGGS J.
Published: November 2003

In vivo studies of lymphocyte biology have used intravenous (i.v.) injection as the primary mode of cell transfer, a protocol consistent with the anatomic distribution of most lymphocytes. However, for study of peritoneal cavity B cells, i.v. injection does not correlate with anatomical localization. This report describes the restoration of B‐cell function in B lymphocyte‐defective X‐chromosome‐linked immune‐defective (XID) mice after intraperitoneal transfer of immunoglobulin heavy chain (Igh)‐disparate peritoneal cavity (PerC) cells. In contrast to i.v. transfer, intraperitoneal (i.p.) transfer restored B‐cell function in young, but not adult (>8 weeks), XID mice. When host and donor Igh allotype matched, PerC B‐cell engraftment was noted in older recipients; this reconstitution however, was also age‐dependent. Migration from the peritoneum to systemic circulation was necessary for serum IgM production as shown by the presence of donor antibody‐secreting cells in the host spleen. Host lymphocytes also influenced the success of i.p. transplantation as severe combined immune‐deficient mice, regardless of age, exhibited donor serum IgM production. Recipient age, Igh allotype, and immune‐deficiency were found to have an impact on the ability of i.p.‐transferred PerC B cells to restore B‐cell function in XID mice.
Complement‐activating ability of leucocytes from patients with‘qc complement factor I deficiency
Authors: MARQUART* H. V. , RASMUSSEN J. M. , LESLIE R. G. Q.
Published: November 2003

Previous studies from this laboratory have shown that normal peripheral blood B cells are capable of activating complement via the alternative pathway (AP), that the activation is associated with complement receptor type 2 (CR2) expression, and that erythrocytes at normal blood levels partially inhibit the activation. The purpose of the present study was to investigate whether factor I (FI) deficiency, which leads to continued formation of the AP convertase (C3bBb) resulting in the consumption of factor B and C3 and large scale generation of C3b fragments, affects the phenotype and/or function of the patients’ B cells. Using flow cytometry, peripheral blood leucocytes (PBL) from two FI‐deficient patients were investigated for expression of complement receptors and complement regulatory proteins, in vivo‐deposited C3 fragments and in vitro complement‐activating ability. CR1 levels on B cells were significantly lower in FI‐deficient patients than in normal individuals, whereas CR2 levels were found to be reduced, although not to a significant extent. CR1 levels on monocytes and polymorphonuclear leucocytes (PMN) were found to be normal or slightly raised. All leucocyte subpopulations were found to be covered in vivo with C3b fragments. AP activation on B cells from FI‐deficient patients in homologous serum was significantly reduced compared with that for normal individuals, whereas no in vitro activation was seen in autologous serum. In addition, the in vivo‐bound C3b fragments were degraded to C3d,g when the patients’ PBL were incubated in homologous serum containing EDTA. Finally, the patients’ erythrocytes failed to exert any inhibition on AP activation in homologous serum.
Functional lipopolysaccharide receptors of low affinity are constitutively expressed on mouse bone marrow cells
Authors: GIRARD R. , PEDRON T. , CHABY R.
Published: November 2003

Although lipopolysaccharide (LPS)‐induced overproduction of cytokines, involved in the pathogenesis of septic shock, occupies the spotlight of endotoxin research, another LPS effect, the differentiation of various cell types including haematopoietic bone marrow cells (BMC), which is probably related to its radioprotective activity, deserves equal attention. We have previously established that nanomolar concentrations of LPS trigger in human BMC the expression of CD14 by an induction mechanism independent of CD14 or any other molecule anchored to the cell membrane by a glycosyl phosphatidylinositol glycolipid. We now show that this LPS‐induced stimulation is triggered by the binding of a small number of LPS molecules (13 000 molecules/cell) to constitutive LPS receptors of low affinity (Kd=480 nm). This interaction, which was inhibited by a synthetic LPS antagonist, appeared specific, reversible, saturable, time‐ and temperature‐dependent, but was independent of divalent cations, and was inhibited by serum. Exposure of BMC to LPS did not induce a down‐modulation of these receptors, but enhanced their sensitivity to trypsin degradation. Inhibition of LPS binding following different treatments correlated with inhibition of BMC stimulation, thus suggesting that the sparse constitutive receptors of low affinity are efficient signalling receptors for LPS.
Variable region structure and Staphylococcal protein A binding specificity of a mouse monoclonal IgM anti‐laminin‐receptor antibody
Authors: FEIJÓ G. C. S. , SABBAGA J. , CARNEIRO C. R. W. , BRÍGIDO M. M.
Published: November 2003

Staphylococcal protein A is a cell wall‐attached polypeptide that acts as a B‐lymphocyte superantigen. This activation correlates with specific VH gene segment usage in the B‐cell receptor. B‐cell receptor assembled from members of the VH3 family in humans, or S107 family in mice, has an intrinsic affinity for protein A. Human VH3‐derived antibodies bind to domain D of protein A. We have characterized a mouse IgM monoclonal antibody that binds protein A. The sequencing of the variable region suggests an almost germline‐encoded VH derived from S107 family and a Vκ8‐derived VL. The binding specificity of the monoclonal antibody was tested with various recombinant constructions derived from protein A. We show that, unlike human VH3‐derived antibody, mouse S107‐derived immunoglobulin binds to the B domain of the bacterial superantigen.
Elimination of T‐cell‐receptor β‐chain diversity in transgenic mice restricts antigen‐specific but not alloreactive responses
Authors: O’BRIEN D. P. , BAECHER‐ALLAN C. M. , BURNS Jr R. P. , SHASTRI N. , BARTH R. K.
Published: November 2003

The contribution of T‐cell‐receptor β‐chain diversity to the T‐cell antigen‐specific repertoire was investigated using single‐chain T‐cell‐receptor transgenic mice. Animals that express the rearranged β‐chain gene from a T hybridoma with specificity for a hen egg lysozyme peptide, designated HEL(85–96) were analysed for their ability to respond to a panel of diverse antigens. Transgenic mice exhibited a significantly elevated response to HEL(85–96) which was shown to be due to an increased frequency of HEL(85–96)‐specific T‐cell progenitors. This increased frequency of specific progenitors resulted in the ability of transgenic mice to respond to the peptide in the absence of antigen priming. Conversely, transgenic mice failed to respond to any other antigen tested. Furthermore, this apparent deficiency was associated with a significant decrease in the frequency of antigen‐specific T‐cell progenitors in transgenic mice. Surprisingly, the ability to launch an alloresponse was unaffected by the exclusive expression of the transgene‐derived β‐chain. These results indicate that β‐chain diversity is crucial for the ability of the T‐cell population to elicit a rapid and robust response to the profusion of different antigen/major histocompatibility complex (MHC) ligands potentially encountered by an individual. Furthermore, these results suggest a lesser role for β‐chain diversity in contributing to allorecognition, and support a model in which the direct recognition of peptide‐mediated conformational MHC forms is the major contributor to the alloreactive response exhibited by the majority of T cells.
Mycobacterium avium infection in mice is associated with time‐related expression of Th1 and Th2 CD4+ T‐lymphocyte response
Authors: AZOUAOU† N. , PETROFSKY M. , YOUNG L. S. , BERMUDEZ L. E.
Published: November 2003

Disseminated infection caused by organisms of Mycobacterium avium complex is common in acquired immune deficiency syndrome (AIDS) patients. M. avium is an intracellular bacterium that multiplies within macrophages. We examined the effect of M. avium infection on the T‐helper cell response in C57/BL/6 black mice. At weekly intervals, CD4+ T‐cells were isolated from spleens and lines were created. T‐cell lines were exposed to sonicated M. avium in the presence of feeder cells and macrophages and the supernatant were collected to measure the concentrations of interferon‐γ (IFN‐γ and interleukin‐10 (IL‐10). Production of IFN‐γ in CD4+ T‐cells obtained from uninfected mice did not vary significantly during the 5 weeks. Levels of IFN‐γ produced by T‐cell lines of infected mice were similar to the control mice during the first 2 weeks but significantly reduced (approximately 30 ng/ml) thereafter. In contrast, production of IL‐10 by T‐cell lines of infected mice was in a range of 190 to 342 pg/ml in weeks 1, 2 and 3, but increased to an average of 1300 pg/ml at weeks 4 and 5. Pre‐immunized mice, when infected with M. avium strain 101, showed a different profile of T‐cell cytokines, with high IFN‐γ and low IL‐10 production. Proteins purified from a number of disease‐associated (D–A) and non‐D–A strains of M. avium were tested for the ability to induce IL‐10. 65 000 MW and 60 000 MW proteins of M. avium induced significantly more IL‐10 than 45 000 MW, 33 000 MW and 27 000 MW proteins. These results showed that M. avium predominantly stimulates either Th1 or Th2 T‐helper cells according to the phase of the infection.
Human γδ T‐cell recognition of Yersinia enterocolitica
Authors: YOUNG J. L. , GOODALL J. C. , BEACOCK‐SHARP H. , HILL GASTON J. S.
Published: November 2003

We have studied the human γδ T‐cell response to Yersinia enterocolitica, a facultative intracellular bacterium which causes gastroenteritis and, particularly in human leucocyte antigen (HLA)‐B27+ individuals, reactive arthritis (ReA). A marked proliferation of that cytotoxic γδ T cells is seen when Yersinia‐infected lymphoblastoid cell lines or fixed intact Yersinia are added to cultures of mononuclear cells derived from the synovial fluid of ReA patients or from the peripheral blood of healthy donors. In contrast, heat‐inactivated Yersinia fail to stimulate the γδ T‐cell response. The γδ T‐cell lines generated killed both autologous and allogeneic infected cell lines. Interestingly, a T‐cell line generated from synovial fluid mononuclear cells (SFMC) killed infected autologous cell lines and a cell line matched for HLA‐B27 less well than infected allogeneic target cells. γδ T‐cell clones isolated from this line were found to express Vγ9Vδ2 T‐cell receptor (TCR) and also killed infected mismatched cells more efficiently than autologous targets. Moreover, from experiments using major histocompatability complex (MHC)‐deficient cell lines, it was apparent that target cell recognition was MHC independent. Our results suggest that γδ T cells can be involved in immunity to Yersinia enterocolitica and should be taken into account when considering immunopathological mechanisms leading to reactive arthritis.
A step‐wise expansion of intestinal intraepithelial T lymphocytes in association with microbial colonization is defined by sensitivity to cyclosporin A
Authors: KAWAGUCHI‐MIYASHITA M. , NANNO M. , SHIMADA S. , NAGAOKA N. , OKADA Y. , MATSUMOTO S. , UMESAKI Y. , MATSUOKA Y. , OHWAKI M.
Published: November 2003

Murine intestinal intraepithelial lymphocytes (IELs) consist of T cells bearing αβ‐antigen receptor (αβ‐IELs) and those bearing γδ‐antigen receptor (γδ‐IELs). Although γδ‐IELs outnumber αβ‐IELs in germ‐free (GF) mice, oral inoculation of fecal suspension from conventional (CV) mice into GF mice induced the increase in number of αβ‐IELs, leaving the number of γδ‐IELs unchanged, and the number of αβ‐IELs reached the level of CV mice by 3 weeks after conventionalization. Expansion of αβ‐IELs and increase in their CD44+ subset in conventionalized mice were not affected until 2 weeks after beginning of daily injection of cyclosporin A (CsA). However, further expansion of αβ‐IELs during 2–3 weeks after conventionalization was blocked by injection of CsA. Although the relative constitution of CD4−8−, CD4+ 8−, CD4−8αα+, CD4−8αβ+ and CD4+ 8+ subsets among αβ‐IELs was comparable between control and CsA‐treated groups, CsA injection resulted in the decrease in ratio of high‐density fraction cells to low density fraction cells in IELs. CsA completely abrogated the expansion of T cells in peripheral lymph nodes stimulated by alloantigens in vivo, and proliferation of IELs from GF mice induced by immobilized anti‐αβ‐T‐cell receptor (TCR) monoclonal antibodies (mAb) in vitro was also eliminated by CsA. These results indicate that microbial colonization‐induced expansion of αβ‐IELs is subdivided into two steps: the early phase of expansion takes place via TCR‐non‐mediated pathway and the late phase of expansion requires TCR‐mediated signal transduction.
Generation of dendritic cells from bone marrow progenitors using GM‐CSF, TNF‐α, and additional cytokines: antagonistic effects of IL‐4 and IFN‐γ and selective involvement of TNF‐α receptor‐1
Authors: LARDON F. , SNOECK H.‐W. , BERNEMAN Z. N. , VAN TENDELOO V. F. I. , NIJS G. , LENJOU M. , HENCKAERTS E. , BOECKXTAENS C. J. , VANDENABEELE P. , KESTENS L. L. , VAN BOCKSTAELE D. R. , VANHAM G. L. E. E.
Published: November 2003

We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two‐stage culture system in which, besides granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and tumour necrosis factor‐α (TNF‐α), stem‐cell factor (SCF) was added during the first 5 days, while interleukin‐4 (IL‐4) and/or interferon‐γ (IFN‐γ) were added during the secondary culture period of 9 days. Addition of IL‐4 favoured the outgrowth of CD1a+, HLA‐DR+, CD4+, CD40+, CD80+ but CD14− cells with dendritic morphology and strong antigen‐presenting capacity. Addition of IFN‐γ selectively induced HLA‐DR and CD86 but did not up‐regulate CD1a expression or antigen‐presenting capacity of the differentiated cells. An antagonism between IL‐4 and IFN‐γ could further be confirmed in that, as compared with IL‐4 alone, the simultaneous addition of IL‐4 and IFN‐γ to GM‐CSF plus TNF‐α during maturation reduced both the phenotypical (CD1a, CD4, CD40) and functional characteristics of DC. Using receptor‐specific TNF‐α mutants, we investigated the relative involvement of TNF‐α receptors R1 and R2 in the generation of DC. The induction of CD1a and HLA‐DR, as well as the increase in allostimulatory capacity were dependent on TNF‐R1 triggering, whereas triggering through TNF‐R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two‐stage culture assay using SCF, GM‐CSF, TNF‐α and IL‐4; second, that the effect of TNF‐α in DC generation involves signalling via the TNF‐R1 receptor; and third, that IFN‐γ counteracts some of the effects of IL‐4 in DC generation.
Expression of major histocompatibility complex antigens on mouse brain microvascular endothelial cells in relation to susceptibility to cerebral malaria
Authors: MONSO‐HINARD C. , LOU J. N. , BEHR C. , JUILLARD P. , GRAU G. E.
Published: November 2003

The physiopathology of experimental cerebral malaria (CM), an acute neurological complication of Plasmodium berghei ANKA (PbA) infection, involves interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α), two cytokines that are known to modulate major histocompatibility complex (MHC) molecule expression. The aim of this study was to evaluate whether the genetic susceptibility to CM is related to the constitutive or IFN‐γ‐induced expression of MHC molecules on brain microvessels. To this end, brain microvascular endothelial cells (B‐MVEC) were isolated from CM‐susceptible (CM‐S, CBA/J) and resistant (CM‐R, BALB/c) mice. By flow cytometry, we found that less than 5% of CM‐S B‐MVEC constitutively expressed MHC class I molecules, in contrast to up to 90% of CM‐R B‐MVEC. Upon stimulation with IFN‐γ, the percentage of positive cells for MHC class I molecules in CM‐S B‐MVEC became comparable to CM‐R B‐MVEC, but a higher fluorescence intensity existed on CM‐S B‐MVEC compared with CM‐R B‐MVEC. MHC class II molecules were not constitutively expressed on B‐MVEC from either strain. IFN‐γ‐induced expression of MHC class II (I‐A, I‐E) molecules was significantly higher in CM‐S than CM‐R B‐MVEC both in percentage of positive cells and fluorescence intensity. These data demonstrate that absent or low MHC class I and higher inducibility of MHC class II expression on B‐MVEC are associated with the genetic susceptibility to CM.
Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1
Authors: BRINKMANN V. , KRISTOFIC C.
Published: November 2003

We have reported previously that uncommitted human CD4+ CD45RO− T cells default to the T‐helper type 1 (Th1) pathway, if they are costimulated by anti‐CD3 plus anti‐CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti‐CD28 plus interleukin‐2 (IL‐2) in the absence of T‐cell receptor (TCR) signals. The anti‐CD28/IL‐2‐induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti‐IL‐4 mAb, suggesting a non‐conventional IL‐4‐independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR Vβ‐chain usage comparable to that of committed Th1 cells (with Vβ8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T‐cell lineage. The induction of cytokine production and B‐cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti‐CD28 or IL‐2. Importantly, anti‐CD28/IL‐2‐primed Th2 cells readily secreted IL‐4 and induced IgE production by surface IgE− B cells in response to the first TCR signal and independent of previous contact with IL‐4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2‐like cells in cancer, infection and allergy is discussed.
Antigen‐specific chemotaxis of B cells
Authors: KOMAI‐KOMA M. , DONACHIE A. M. , WILKINSON P. C.
Published: November 2003

The chemoattractant effect of soluble protein antigens for B cells from immunized mice was examined. Mice were immunized either via the footpad with ovalbumin (OVA) in complete Freund’s adjuvant (CFA) or with CFA alone; or intraperitoneally with OVA incorporated in immune‐stimulating complexes (OVA–ISCOM) or with saline. After 8–14 days B cells were purified from the spleens or the draining popliteal nodes and tested in vitro for locomotor responses to antigen using polarization (shape‐change) and filter assays. Cells obtained by both routes of immunization, but not cells from control mice, gave locomotor responses to OVA. Responses were seen in B cells directly after preparation (5–8% of cells responding) but were enhanced if the cells were cultured overnight in the presence of interleukin‐4 (IL‐4) before testing (10–12% of cells responding). Checkerboard filter assays suggested that the response to OVA was chemotactic. The response was antigen specific since cells from OVA‐immunized mice did not respond to bovine serum albumin (BSA), and cells from BSA‐immunized mice responded to BSA but not to OVA. The response to OVA was inhibited by preincubation of OVA with anti‐OVA but not with anti‐BSA. Many of the cells that polarized in response to antigen were larger than any B cells found in control populations suggesting that the responsive cells are those that had been stimulated to enter cell cycle following immunization.
TCR αβ+ CD4− CD8− T cells differentiate extrathymically in an lck‐independent manner and participate in early response against Listeria monocytogenes infection through interferon‐γ production
Authors: KADENA T. , MATSUZAKI G. , FUJISE S. , KISHIHARA K. , TAKIMOTO H. , SASAKI M. , BEPPU M. , NAKAMURA S. , NOMOTO K.
Published: November 2003

T‐cell receptor (TCR) αβ+ CD4− CD8− (double‐negative; DN) T cells appear in the peritoneal cavity at an early stage of intraperitoneal (i.p.) infection with the intracellular pathogen Listeria monocytogenes. In the present report, we analysed the developmental pathway and functions of the TCRαβ+ DN T cells using the L. monocytogenes infection system. The TCRαβ+ DN T cells appeared in the peritoneal cavity after L. monocytogenes i.p. infection in adult‐thymectomized lethally irradiated bone marrow chimeras and p56lck‐deficient mice. The results demonstrated that the TCRαβ+ DN T cells can develop extrathymically in a p56lck‐independent manner. Reverse transcription–polymerase chain reaction (RT‐PCR) analysis showed that the TCRαβ+ DN T cells expressed genes for interferon‐γ (IFN‐γ), the macrophage chemotactic factors MCP‐1 and Eta‐1, and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) but lacked expression of genes for interleukin‐2 (IL‐2), IL‐4 and IL‐10. As expected from the RT‐PCR analysis, the TCRαβ+ DN T cells produced IFN‐γ in response to anti‐TCRβ monoclonal antibody (mAb), anti‐CD3 mAb and L. monocytogenes‐infected macrophages but IL‐4 was undetectable after the stimulation. Furthermore, the intracellular cytokine staining analysis demonstrated that approximately half of the TCRαβ+ DN T cells detectable at the early stage of L. monocytogenes infection were IFN‐γ‐producing cells. All of the results suggest that the TCRαβ+ DN T cells develop through a unique extrathymic p56lck‐independent pathway and participate in early protection against bacterial infection through activation and accumulation of macrophages.
The characterization of testicular cell (TC)‐specific T‐cell clones induced by intratesticular Listeria monocytogenes infection: TC‐specific T cells with atypical cytokine profile transfer orchitis
Authors: MATSUZAKI G. , SONODA K.‐H. , MUKASA A. , YAMADA H. , NAKAMURA T. , IKEBE H. , HAMANO S. , NOMOTO K.
Published: November 2003

A unilateral infection of Listeria monocytogenes into the testis of mice induces not only Listeria‐specific T cells but also autoreactive T cells that can transfer experimental autoimmune orchitis (EAO) into naive mice. To investigate the characteristics of the autoreactive T cells, we established six testicular cell (TC)‐specific T‐cell clones from the spleen of the intratesticularly infected mice. All the clones expressed CD4 and T‐cell receptor (TCR) αβ, and four of the six clones expressed Vβ8. They showed proliferative response to TC in the presence of syngeneic spleen antigen‐presenting cells, but did not cross‐react to Listeria antigen (Ag). They produced interferon‐γ (IFN‐γ) when stimulated with TC, but interleukin‐2 (IL‐2), IL‐4 and IL‐10 were undetectable. IL‐2 production was not detected even when they were restimulated with TC after a 10‐day resting culture without Ag and IL‐2, although they proliferated in the restimulation culture. Even in the presence of anti‐IL‐2 mAb, the TC‐specific T‐cell clones showed proliferative response against TC. The observations indicate that the TC‐specific IFN‐γ‐producing T cells proliferate in the absence of autocrine. Both intravenous and intratesticular injection of these clones transferred EAO in syngeneic naive mice. These results suggest that L. monocytogenes infection in the testis induces autoreactive orchitogenic CD4+ T cells without cross‐reactivity to bacterial Ag. Furthermore, these data demonstrate that CD4+ T cells with an atypical cytokine profile can efficiently cause EAO.
Regulation of experimental autoimmune orchitis by the presence or absence of testicular antigens during immunological development in SCID mice reconstituted with fetal liver cells
Authors: WAKABAYASHI A. , EISHI Y. , NAKAMURA K.
Published: November 2003

Severe combined immunodeficient (SCID) mice were immunologically reconstituted by the transfer of fetal liver cells (FLC) of BALB/c mice (SCID‐FLC mice). In peripheral blood (PB) of SCID‐FLC mice, B and T cells started to appear 2 and 5 weeks, respectively, after the transfer of FLC, and had attained normal levels by 7 weeks. Orchidectomy and transplantation of testis under the kidney capsule were conducted at various stages of immunological maturation, and the induction of experimental autoimmune orchitis (EAO) was performed after immunological maturation in SCID‐FLC mice. The experimental system was used to establish that the presence of testicular antigens in the early stage of immunological development influences the induction of EAO; grade of EAO was reduced in the presence of the antigens, and enhanced in their absence. In other words, the existence of self tissue antigens in the early stage of immunological development was essential for proper establishment of tolerance to the self tissue. These findings suggested that the SCID‐FLC mouse is a suitable model with which to analyse the interaction between self antigens and cells of the developing immune system, which is otherwise observed only in the fetal or perinatal stage in experimental animals.
A multimeric form of soluble recombinant sheep LFA‐3 (CD58) inhibits human T‐cell proliferation
Authors: YAMASHITA K. , PARISH C. R. , WARREN H. S. , HARRISON L. C.
Published: November 2003

The rosetting of T cells by sheep erythrocytes is mediated through the interaction of the CD2 molecule on T cells with T11TS, a molecule on sheep erythrocytes homologous to lymphocyte function‐associated antigen‐3 (LFA‐3, CD58). We cloned a T11TS cDNA from sheep leucocyte mRNA which encodes a soluble molecule comprising the distal D1 and the D2 extracellular domains, but not the transmembrane domain. cDNA for this soluble D1+D2 form of sheep LFA‐3 (sLFA‐3) was expressed in Escherichia coli and the properties of the purified recombinant protein were assessed by inhibition of T‐cell rosette formation. sLFA‐3 inhibited rosette formation, but its activity was low, 50% inhibition occurring at 25 μg/ml, consistent with the observed low binding avidity of fluorescein isothiocyanate (FITC)‐labelled sLFA‐3. sLFA‐3 was made multimeric to increase its affinity, by crosslinking biotinylated sLFA‐3 to streptavidin–biotinylated dextran complexes. The binding of crosslinked sLFA‐3 multimers, tested by fluorescence‐activated cell sorting (FACS) analysis, was significantly increased compared to sLFA‐3 monomers. Competition with monoclonal antibodies demonstrated that multimeric sLFA‐3 bound to the T111 epitope on CD2. The multimeric form of sLFA‐3 was significantly more potent than the monomer in inhibiting proliferation of human T cells in response to purified protein derivative (PPD), tetanus toxoid (TT) or allogeneic cells. Multimeric sLFA‐3 might, therefore, have potential as an immunotherapeutic agent to inhibit and/or anergize antigen‐specific T‐cell responses.
Regulation of cytokine production by human Th0 cells following stimulation with peptide analogues: differential expression of TGF‐β in activation and anergy
Authors: TSITOURA D. C. , GELDER C. M. , KEMENY D. M. , LAMB J. R.
Published: November 2003

The different biological activities of T‐cell‐derived cytokines and their level of production influences the qualitative nature of immune responses and, in certain forms of T‐cell tolerance, the lack of antigen responsiveness is associated with the production of transforming growth factor‐&bgr; (TGF‐&bgr;) and interleukin‐4 (IL‐4). In this study we have investigated the effects of T‐cell receptor (TCR) ligation with peptide analogues and the native peptide, in the presence and absence of costimulation, on cytokine production by human T‐helper type 0 (Th0) cells reactive with influenza virus haemagglutinin (HA) peptide (HA306–318) and restricted by HLA‐DRB1*0101. We observed that resting Th0 cells constitutively produced TGF‐&bgr;, but when stimulated with peptide and antigen‐presenting cells (APC) under conditions that induce clonal expansion, TGF‐&bgr; secretion was abrogated. Furthermore, exposure of the T cells to the wild‐type HA peptide under conditions that induce T‐cell anergy resulted in the secretion of TGF‐&bgr;, and subsequent antigenic rechallenge was unable to override this signal and down‐regulate TGF‐&bgr; production. Stimulation with altered TCR ligands that failed to induce proliferation also resulted in marked production of TGF‐&bgr;, although in many instances the levels were less than those observed in the total absence of antigen, suggesting that partial signalling has occurred. Although in general, there was a direct positive correlation between proliferation and the production of IL‐2, IL‐4 and interferon‐&ggr; (IFN‐&ggr;) following stimulation with certain analogues, the production of selected cytokines was dissociated.
Expression of the neonatal Fc receptor, FcRn, on human intestinal epithelial cells
Authors: ISRAEL E. J. , TAYLOR S. , WU Z. , MIZOGUCHI E. , BLUMBERG R. S. , BHAN A. , SIMISTER N. E.
Published: November 2003

Maternal IgG is transferred to the suckling mouse and rat through a major histocompatibility complex (MHC) class I‐related Fc receptor (FcRn) on the brush border of the proximal small intestine. We have previously described a site on the epithelial surface of the human fetal intestine with IgG binding characteristics similar to FcRn. We report here the identification by reverse transcriptase polymerase chain reaction amplification and sequencing of the human orthologue of rat and mouse FcRn in tissue obtained from human fetal and adult intestine. FcRn protein was detected in adult human intestine by western blot. Immunohistochemical studies of sections of human intestine show that the FcRn is localized mostly to the epithelial cells, where it is in the apical region. These data suggest that the binding of IgG previously seen in the fetal intestine is due to the presence of FcRn. Potential roles for this MHC class I‐like Fc receptor in the human intestine include the transfer of passive immunity, induction of oral tolerance, and immunosurveillance.
Cellular distribution of a natural killer cell tumour recognition‐related surface antigen in purified human lymphocytes
Authors: ALKHATIB G. , MURATA K. , RODER J. C.
Published: November 2003

Natural killer (NK) cells are large granular lymphocytes capable of human leucocyte antigen (HLA) unrestricted killing of tumour cells. A putative NK cell tumour‐recognition molecule (NK‐TR) was previously isolated and cloned. The predicted primary structure of the NK‐TR revealed that the amino terminus of the protein shared high homology with cyclophilin proteins. In this study, we used rabbit antibodies directed against synthetic peptides corresponding to amino acids 476–497 of the NK‐TR protein, to examine the expression of the NK‐TR antigen in freshly purified human lymphocytes. Cell‐surface staining experiments using these peptide antibodies indicated the presence of the NK‐TR protein on the surface of human CD3+ T‐cell populations purified from peripheral blood. There were individual donor differences in the levels of cell‐surface expression of this antigen ranging from 35 to 90% in T lymphocytes and NK cells purified from different healthy volunteers. The immunoreactivity of our peptide antibodies in immunoprecipitation showed that the NK‐TR‐related protein expressed in purified T cells is similar to that expressed in NK cells in terms of its electrophoretic mobility. Cell‐surface staining experiments using the peptide antibodies revealed that the NK‐TR‐related protein is more abundantly expressed on the surface of purified T cells compared with NK cells. Northern blot analysis of the mRNA species transcribed in human lymphocytes revealed abundant expression of NK‐TR‐specific mRNA species in purified T cells. Furthermore, another mRNA species smaller than 7 kb was detected in both NK and T‐cell populations of lymphocytes freshly isolated from peripheral blood. Expression at the cell surface of a cyclophilin‐homologous protein in purified human T lymphocytes may indicate another function for the reported NK‐TR protein, that is, distinct from tumour‐cell recognition and cytosis.
Adhesion molecules are upregulated on dendritic cells isolated from human blood
Authors: McCARTHY D. A. , MACEY M. G. , BEDFORD P. A. , KNIGHT S. C. , DUMONDE D. C. , BROWN K. A.
Published: November 2003

This study investigated whether the high expression of adhesion molecules on enriched preparations of circulating dendritic cells (DCs) was an intrinsic property of the cells or whether it was a consequence of the procedure used to isolate them from blood. Expression of the &bgr;1, &bgr;2 integrins (CD11/CD18 family) and other adhesion molecules on DCs in whole blood was compared with that on isolated DCs. Dendritic cells were identified by flow cytometry as leucocytes that were positive for human leucocyte antigen (HLA)‐DR, but negative for CD3, CD14, CD16, CD19 and CD56. In contrast to a minority of DCs in whole blood, the majority of isolated DCs expressed the &bgr;2 integrins and there were a greater number of cells bearing CD44, CD54 and some of the &bgr;1 integrins (notably CD49b, CD49d, CD49e and CD29). An increase in the proportion of DCs bearing adhesion molecules was generally apparent at the isolation stage when mononuclear cells, which had been incubated overnight, were centrifuged on a metrizamide gradient to enrich for cells of low density. Inclusion of an inhibitor of protein glycosylation and exocytosis (brefeldin A) at all stages of separation partially prevented an increase in the percentage of DCs bearing CD18, C29 and C54 whereas the inclusion of cycloheximide (an inhibitor of polypeptide synthesis) interfered with increases in the percentage of cells bearing CD29 and CD54. Neither of these antagonists had an effect on the intensity of adhesion molecule expression. We suggest that some of the adhesion‐dependent functions of isolated DCs are caused, in part, by an upregulation of surface adhesion molecules induced by the enrichment procedure.