bluuebunny/crossref_metadata_2025 huggingface dataset:
VCAM‐1 contributes to rapid eosinophil accumulation induced by the chemoattractants PAF and LTB4: evidence for basal expression of functional VCAM‐1 in rat skin
Authors: DAVIES¶  , LARBI  , ALLEN  , SANZ  , WEG  , HASKARD  , LOBB  , NOURSHARGH
Published: March 2003

The aim of the present study was to investigate the role of the adhesion pathway α4 integrins/vascular cell adhesion molecule type 1 (VCAM‐1) in rapid eosinophil accumulation induced by the chemoattractants PAF and LTB4. For this purpose we have used an in vivo model of local 111In‐eosinophil accumulation to quantify eosinophil accumulation induced by intradermal administration of platelet‐activating factor (PAF) and leukotriene B4 (LTB4) in rats. Initial experiments carried out over 4 hr demonstrated that intravenous administration of an anti‐VCAM‐1 monoclonal antibody (mAb; 5F10) or an anti‐α4 integrin mAb (TA2) caused a significant reduction in PAF‐ or LTB4‐induced 111In‐labelled eosinophil accumulation. Time–course experiments demonstrated that the anti‐VCAM‐1 mAb was effective at suppressing early phases of the 111In‐labelled eosinophil accumulation induced by PAF and LTB4 (e.g. within the first 60 min). In contrast, 111In‐labelled eosinophil accumulation induced by these chemoattractantswas unaffected by the local administration of the transcriptional inhibitor actinomycin D, suggesting a role for basally expressed VCAM‐1. Indeed, basal expression of VCAM‐1 in rat skin sites was demonstrated by the localization of intravenously administered radiolabelled mAb. The localization of the radiolabelled antibody was not altered in skin sites injected with PAF or LTB4. Finally, the inhibitory effects seen with the anti‐VCAM‐1 mAb were enhanced when the antibody was co‐injected into rats with an anti‐intercellular adhesion molecule‐1 (ICAM‐1) mAb (1A29). The combination of these two mAb also caused a significant inhibition of PAF‐induced oedema, as quantified by the local accumulation of 125I‐labelled human serum albumin. The results indicate a role for α4 integrins/VCAM‐1 and ICAM‐1, in PAF‐ and LTB4‐induced eosinophil accumulation in vivo and suggest that basally expressed VCAM‐1 may have a functional role in rapid accumulation of eosinophils induced by chemoattractants.
In vivo microscopy of murine islets of Langerhans: increased adhesion of transferred lymphocytes to islets depends on macrophage‐derived cytokines in a model of organ‐specific insulitis
Authors: Ludwig  , Kretschmer  , Caspar  , Bojunga  , Oldenburg  , Schumm‐Draeger  , Stegmüller  , Von Minckwitz  , Usadel  , Kusterer
Published: March 2003

Environmental factors contribute to the pathogenesis of type 1 diabetes (insulin‐dependent diabetes mellitus). Multiple low doses of streptozotocin (MLDS) induce hyperglycaemia and insulitis in mice. Previously we demonstrated that adhesion of lymphocytes to endothelium of islets is only increased when donor animals were diabetic and recipient mice had received 5 mg/kg streptozotocin (STZ). Therefore we used streptozotocin to evaluate the immunological relevance of such an irritation of islets. Lymphocytes, separated from diabetic mice (MLDS), were fluorescently labelled and injected to recipient mice that had received 5 mg/kg STZ. With in vivo microscopy we measured lymphocyte flow and adherence in islets. Expression of vascular cell adhesion molecule‐1 (VCAM‐1) and intracellular adhesion molecule‐1 (ICAM‐1) in the pancreas was assessed using immunohistochemistry. Very late antigen‐4 (VLA‐4) and leucocyte function‐associated antigen‐1 (LFA‐1) expression on transferred lymphocytes was measured with flow cytometry. Pretreatment of recipients with antibodies to cytokines or silica reduced lymphocyte adherence to islet endothelium from 2·04% (goat immunoglobulin G; IgG) or 1·82% (rat IgG) to 0·47, 0·58, 0·39 or 0·19% for monoclonal antibody (mAb) interferon‐γ (IFN‐γ), polyclonal antibody (pAb) tumour necrosis factor‐α (TNF‐α), pAb interleukin (IL)‐1α or silica, respectively. Reduced adhesion was associated with a decreased expression of VCAM‐1 and ICAM‐1 in islets of treated recipients compared with mice treated with 5 mg/kg STZ alone. In conclusion, pretreatment of recipients with 5 mg/kg STZ leads to an increased expression of adhesion molecules in the islets and lymphocyte adhesion to islet endothelium in vivo, demonstrating an immune response of the islets. Prevention of increased expression of ICAM‐1 or VCAM‐1 and reduction of lymphocyte adhesion in islets by silica or antibody indicate an involvement of macrophages and macrophage derived cytokines in the generation of this immune response.
Milk cytokines and subclinical breast inflammation in Tanzanian women: effects of dietary red palm oil or sunflower oil supplementation
Authors: Filteau  , Lietz  , Mulokozi G. , Bilotta S. , Henry  , Tomkins
Published: March 2003

Previously, we have found that subclinical breast inflammation, as indicated by raised breastmilk concentrations of sodium and the inflammatory cytokine, interleukin‐8 (IL‐8), was highly prevalent in Bangladesh and associated with poor infant growth. In order to investigate further the prevalence of subclinical breast inflammation and to assess the impact of dietary intervention, we studied rural Tanzanian women taking part in a study of dietary sunflower or red palm oil supplementation during late pregnancy and lactation. We measured breastmilk concentrations of IL‐8, the anti‐inflammatory cytokine, transforming growth factor‐β2 (TGF‐β) and the ratio of sodium to potassium. We also estimated systemic inflammation by plasma concentrations of the acute phase proteins, α1‐acid glycoprotein and C‐reactive protein. There were highly significant intercorrelations among milk Na/K ratio and concentrations of IL‐8 and TGF‐β, the last only after treatment with bile salts which also improved TGF‐β recovery in the enzyme‐linked immunosorbent assay (ELISA). Plasma acute phase protein concentrations tended to correlate with milk Na/K ratio and IL‐8, suggesting that subclinical breast inflammation was related to systemic inflammation. Dietary supplementation with vitamin E‐rich sunflower oil but not provitamin A‐containing red palm oil decreased milk Na/K, IL‐8 and TGF‐β at 3 months postpartum; however, the effect was significant only for Na/K ratio. The results suggest that milk Na/K ratio, IL‐8, and TGF‐β all measure the same phenomenon of subclinical breast inflammation but that Na/K ratio, having the lowest assay variability, is the most useful. Subclinical breast inflammation may result in part from systemic inflammation and may be improved by increased dietary intake of vitamin E‐rich sunflower oil.
CD40‐deficient dendritic cells producing interleukin‐10, but not interleukin‐12, induce T‐cell hyporesponsiveness in vitro and prevent acute allograft rejection
Authors: Gao  , Madrenas  , Zeng  , Cameron  , Zhang  , Wang  , Zhong  , Grant
Published: March 2003

The induction of an immune response or tolerance is mediated by corresponding subsets of dendritic cells (DC). However, the property of tolerogenic DC is not clear. Recently, we have characterized a population of CD11c+ splenic DC derived from long‐term mixed leucocyte culture (LT‐MLC), which are able to proliferate upon stimulation and have a strong primary mixed leucocyte reaction (MLR)‐stimulating activity in conventional MLR. In this study, we show that, in contrast to the irradiated ones, non‐irradiated LT‐MLC‐derived DC induce polyclonal antigen‐specific T‐cell hyporesponsiveness when cocultured with allogeneic splenocytes for 3–11 days. The degree of the hyporesponsiveness increased with the length of coculture. Although these DC expressed major histocompatibility complex class II and B7 costimulatory molecules, which are down‐regulated during coculture, they expressed very low or undetectable CD40 before and after coculture, respectively. The CD40‐deficient DC spontaneously produce interleukin‐10 (IL‐10), but not IL‐12. The skewed balance between IL‐10 and IL‐12 is associated with their capability to induce T‐cell hyporesponsiveness, because a neutralizing antibody to IL‐10, exogenous recombinant IL‐12 or lipopolysaccharide (LPS) significantly blocked the hyporesponsiveness. Accordingly, infusion of a small number of non‐irradiated LT‐MLC‐derived DC (5×105) significantly prolonged the survival of a vascularized heterotopic murine heart transplant, whereas irradiated DC accelerated graft rejection. These data suggest that CD40‐deficient DC producing IL‐10, but not IL‐12 can induce T‐cell hyporesponsiveness in vitro and in vivo.
T‐cell recognition of lipid peroxidation products breaks tolerance to self proteins
Authors: Wuttge  , Bruzelius  , Stemme
Published: March 2003

Peroxidation of polyunsaturated fatty acids in lipoproteins and cell membrane phospholipids occurs in many situations in the body, both under normal and pathological conditions. Low‐density lipoprotein is particularly prone to oxidation and is believed to be a pathogenetic component in atherogenesis. Both antibody responses and T‐cell responses to oxidatively modified lipoproteins have been demonstrated in humans as well as in animal models. However, little is known about how these responses arise or how T cells recognize these antigens. In the present study, mice were immunized with homologous albumin covalently modified with a series of defined aldehydes which are known to be generated during lipid peroxidation. T‐cell hybridomas from immunized animals demonstrated major histocompatibility complex‐restricted and protein sequence‐dependent responses to modified albumin, but not to native albumin. In addition to the response to modified epitopes, some aldehyde modifications resulted in strong antibody responses also to the non‐modified protein. This T‐cell‐dependent break of tolerance constitutes a novel pathway for induction of autoimmunity by lipid peroxidation. The findings have implications in many situations where lipid peroxidation products are generated, including atherosclerosis and inflammatory and infectious diseases.
Contribution of mast cells to the T helper 2 response induced by simultaneous subcutaneous and oral immunization
Authors: Aoki I. , Itoh S. , Yokota S. , Tanaka S.‐I. , Ishii N. , Okuda K. , Minami M. , Klinman D. M.
Published: March 2003

This work examines the contribution of mast cells to the synergistic enhancement of the T helper 2 (Th2) immune response elicited following simultaneous oral and subcutaneous (s.c.) immunization. The s.c. route induced a Th1‐biased immune response, characterized by increased interferon‐γ (IFN‐γ) and immunoglobulin G2a (IgG2a) antibody production. In contrast, oral immunization stimulated a primarily Th2‐type response in which interleukin‐4 (IL‐4) and IgG1 antibody production were dominant. Simultaneous immunization also triggered a Th2‐biased response, the magnitude of which exceeded the additive effects of s.c. and oral immunization alone by greater than threefold. To analyse whether mast cells in gut‐associated lymphoid tissue contributed to this synergistic response, mast cell‐deficient mice WBB6F1‐w/wv were studied. Whereas the primary response following simultaneously antigen administration was reduced only twofold in these animals compared with wild type controls WBB6F1‐+/+ (suggesting that mast cells were not needed to initiate Th2 immunity), reconstitution with bone‐marrow‐derived mast cells from WBB6F1‐+/+ mice resulted in a superoptimal response (suggesting that mast cells contribute to the magnitude and perpetuation of these Th2‐biased responses).
l‐selectin expression on thymic emigrants defines two distinct tissue‐migration pathways
Authors: Holder J. E. , Kimpton W. G. , Washington E. A. , Cahill R. N. P.
Published: March 2003

We have studied the appearance and phenotype of recent thymic emigrants in blood, spleen and lymph nodes (LN) of neonatal lambs. Using in situ labelling of thymocytes with fluoroscein isothiocyanate (FITC), we examined the expression of the LN homing receptor
l‐selectin on αβ and γδ subsets of recent thymic emigrants 24 hr after labelling. There were marked differences in the proportions of CD4+, CD8+ and γδ T‐cell receptor (TCR+) cells exported from the thymus to spleen compared to lymph nodes. Spleen was enriched in CD8+ and γδ TCR+ emigrants while LN were enriched in CD4+ emigrants. There were also marked differences in the expression of
l‐selectin by emigrants homing to spleen compared with those homing to lymph nodes. While the majority of thymic emigrants in LN expressed
l‐selectin, considerably fewer emigrants in spleen were
l‐selectin+. The presence of large numbers of CD8+ l‐selectin– and γδ TCR+ l‐selectin– thymic emigrants homing to spleen raises the possibility that unique homing receptor specificities underpin the migration of T cells to spleen as distinct from lymph nodes.
Identification of cation‐independent mannose 6‐phosphate receptor/insulin‐like growth factor type‐2 receptor as a novel target of autoantibodies
Authors: Tarrago D. , Aguilera I. , Melero J. , Wichmann I. , Nuñez‐roldan A. , Sanchez B.
Published: March 2003

Two human monoclonal autoantibodies, B‐33 and B‐24, were generated from the B cells of a patient with scleroderma. Both monoclonal antibodies (mAbs) were composed of µ and λ chains, and recognized cytoplasmic vesicular structures by indirect immunofluorescence on Hep‐2 cell line slides, although mAb B‐24 showed an additional diffuse cytoplasmic staining pattern. By Western blot, mAb B‐24 exhibited a polyreactive‐like binding pattern, whereas mAb B‐33 failed to recognize any electroblotted Hep‐2 antigen. The polyreactive versus monospecific behaviour of mAbs B‐24 and B‐33 was further confirmed by enzyme‐linked immunosorbent assay (ELISA) with a variety of foreign and autoantigens. The N‐terminal sequence of a protein band isolated by affinity chromatography with mAb B‐33 was identical to that of cation‐independent mannose 6‐phosphate receptor (CI‐MPR), also known as the insulin‐like growth factor type‐2 receptor (IGF‐2R). Immunofluorescence experiments on Hep‐2 cell line slides demonstrated a striking co‐localization between the staining pattern exhibited by these mAbs and the pattern obtained using a goat anti‐CI‐MPR serum, indicating the recognition by B‐24 and B‐33 of a structure located predominantly in late endosomes. Sequence analysis of the V‐region gene segments of B‐33 and B‐24 showed both to be identical, except for the existence of a point mutation in B‐33 located in the H‐complementarity‐determining region 3 (H‐CDR3) (position 100D), which produces a non‐conservative replacement of Gly by Ser. This single replacement appears to be responsible for the dramatic change in reactivity of human mAb B‐33. The data shown here provide new evidence of the critical role played by the H‐CDR3 region in distinguishing a polyspecific from a monospecific antibody. A population study demonstrated the existence of immunoglobulin G (IgG) reactivity against CI‐MPR/IGF‐2R in serum specimens from five individuals with different pathological conditions, thus indicating that this molecule is a potential target for the human autoimmune response.
Large clonal expansions of human virus‐specific memory cytotoxic T lymphocytes within the CD57+ CD28– CD8+ T‐cell population
Authors: Weekes M. P. , Wills M. R. , Mynard K. , Hicks R. , Sissons J. G. P. , Carmichael A. J.
Published: March 2003

The proportion of human peripheral blood CD8+ T cells that are CD57+ CD28– is low at birth but increases with age and in individuals infected with human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV). These CD57+ CD28– CD8+ T cells contain large oligoclonal T‐cell expansions whose antigen specificity is unknown. We identified clonal expansions of virus‐specific memory cytotoxic T‐lymphocyte precursors (CTLp) in both healthy carriers of HCMV and in asymptomatic HIV‐infected subjects. In each subject, from the T‐cell receptor (TCR) β‐chain hypervariable sequence of each immunodominant CTL clone, we designed complementary oligonucleotide probes to quantify the size and phenotypic segregation of individual virus‐specific CTL clones in highly purified populations of peripheral blood CD8+ T cells. We found large clonal expansions of virus‐specific CTL clonotypes in CD57+ CD28– CD8+ T cells. Using limiting dilution analysis, we found functional peptide‐specific CTLp at high frequency in CD57+ CD28– cells. Thus, memory CTL specific for persistent viruses account for many oligoclonal expansions within CD57+ CD28– CD8+ T cells.
Naturally occurring polyphenolic antioxidants modulate IgE‐mediated mast cell activation
Authors: Chen S.‐S. , Gong J. , Liu F.‐T. , Mohammed U.
Published: March 2003

Reactive oxygen species (ROS) are known to modulate activities of a host of kinases, phosphatases and transcription factors. Rutin and chlorogenic acid (CGA) are the major polyphenolic antioxidants present in the small molecular fraction of smokeless tobacco leaf extracts, as ascertained by reverse‐phase high‐pressure liquid chromatography (HPLC) and mass spectrometry. Levels of intracellular ROS in resting versus antigen–immunoglobulin E (IgE)‐challenged murine mast cells were measured at 510 nm by fluorescence‐activated cell sorting (FACS) using carboxy‐dichlorofluorescein (DCFH‐DA). Enhanced ROS production was observed in IgE‐sensitized mast cells following antigenic challenge. Rutin and CGA reduced ROS levels in antigen–IgE‐activated mast cells. Concomitantly, they also profoundly inhibited histamine release by these activated mast cells. In contrast, rutin and CGA augmented the inducible cytokine messages, i.e. interleukin (IL)‐10, IL‐13, interferon‐γ (IFN‐γ), IL‐6 and tumour necrosis factor‐α (TNF‐α) in IgE‐sensitized mast cells following antigen challenge. This study indicates that tobacco polyphenolic antioxidants that quench intracellular ROS, differentially affect two effector functions of antigen–IgE‐activated mast cells. This model system may be employed to determine the molecular target of polyphenols. The potential role of these polyphenolic antioxidants on IgE‐mediated allergy in vivo depends on a balance of their differential effects on mast cell activation.
Induction of functional CD154 (CD40 ligand) in neonatal T cells by cAMP‐elevating agents
Authors: Suárez A. , Mozo L. , Gayo A. , Simó A. , Gutiérrez C.
Published: March 2003

A deficiency of neonatal T lymphocytes to express CD154 antigen in response to ionomycin and phorbol 12‐myrsistate 13‐acetate (PMA) stimulation or after CD3 cross‐linking has been described. In the present report we describe that CD45RA+ newborn cells are able to synthesize and express CD154 at similar or even higher levels than adult cells in response to ionomycin and cAMP‐elevating agents which trigger the protein kinase A (PKA) ‐mediated metabolic pathway. Peak CD154 protein concentrations in newborn cells were found between 4 and 8 hr after stimulation with ionomycin and dibutyryl cAMP. These agents, however, did not induce expression of the early activation antigen CD69. Surface levels of CD154 did not correlate with specific mRNA concentration, indicating that dibutyryl cAMP up‐regulates CD154 by acting at a post‐transcriptional stage. The CD154 antigen induced by PKA activation of newborn cells was functional, since upon binding to CD40 on B lymphocytes in the presence of interleukin‐4 (IL‐4), it promoted immunoglobulin heavy‐class switching to IgE. We also found a different pattern of cytokine production between neonatal and adult CD4+ T cells. In response to ionomycin and dibutyryl cAMP, cord blood cells were more prone than adult lymphocytes to secrete the T helper type 2‐derived immunosuppressive cytokines IL‐4 and IL‐10. Taking into account that the feto–maternal environment is rich in cAMP‐elevating agents, the reduced risk of graft versus host disease associated with cord blood trasplantation, as compared with the risk with adult bone marrow cell transplants, may be due to the bias of neonatal cells to differentiate towards the T helper type 2 functional cell subset.
Failure to remove autoreactive Vβ6+ T cells in Mls‐1a newborn mice attributed to the delayed development of B cells in the thymus
Authors: Touma M. , Mori K. J. , Hosono M.
Published: March 2003

Clonal deletion of autoreactive T cells in the thymus is one of the major mechanisms for establishing tolerance to self‐antigens, and self‐reactive T cells bearing Vβ6 T‐cell receptors are usually deleted before their maturation in Mls‐1a mice. However, these T cells develop transiently in the neonatal thymus, and migrate to the periphery. In order to understand the mechanisms which permit these potentially auto‐toxic T cells to generate, we investigated in vivo the physiological or functional properties of the elements involved, such as neonatal T cells, antigens and antigen‐presenting cells (APC). Confirming the previous findings that each of these elements per se is already completed in function in neonates, we investigated the possibility of the absence or immaturity of particular APC with Mls antigens of their own products in the neonatal thymus. In the search for the cellular and histological changes occurring in the newborn thymus, we found that the elimination of Vβ6+ T cells progressed in parallel with the development of thymic B cells. Involvement of B cells in purging the autoreactive T cells from the newborn thymus was shown by prevention of the deletion of Vβ6+ T cells after the removal of B cells by the treatment of neonates with anti‐immunoglobulin M antibodies. The restricted and stable expression of CD5 on the thymic B cells, but not on the splenic cells, suggests that these B cells are not postnatal immigrants from the periphery. Finally, it is concluded that the deficiency in the deletion of self‐reactive T cells in the thymus of Mls‐1a neonates is due to the delayed development of B cells.
Vitamin E supplementation increases T helper 1 cytokine production in old mice infected with influenza virus
Authors: Han S. N. , Wu D. , Ha W. K. , Beharka A. , Smith D. E. , Bender B. S. , Meydani S. N.
Published: March 2003

Compared with young mice, old mice infected with influenza virus have significantly higher pulmonary viral titres, although these can be reduced significantly with dietary vitamin E supplementation. T helper 1 (Th1) cytokines, especially interferon‐γ (IFN‐γ), play an important role in defending against influenza infection. However, there is an age‐associated loss of Th1 cytokine production. Prostaglandin E2 (PGE2) production, which increases with age, can modulate the T helper cell function by suppressing Th1 cytokine production. To investigate the mechanism of vitamin E supplementation on reduction of influenza severity in old mice, we studied the cytokine production by splenocytes, and PGE2 production by macrophages (Mφ), in young and old C57BL mice fed semipurified diets containing 30 (control) or 500 parts per million (ppm) (supplemented) vitamin E for 8 weeks, and then infected with influenza A/PC/1/73 (H3N2). Old mice fed the control diet had significantly higher viral titres than young mice; old mice fed the vitamin E‐supplemented diet had significantly lower pulmonary viral titres than those fed the control diet (P = 0·02 and 0·001 for overall age and diet effect, respectively). Following influenza infection, interleukin (IL)‐2 and IFN‐γ production was significantly lower in old mice than in young mice. Vitamin E supplementation increased production of IL‐2 and IFN‐γ in old mice; higher IFN‐γ production was associated with lower pulmonary viral titre. Old mice fed the control diet showed significantly higher lipopolysaccharide (LPS)‐stimulated Mφ PGE2 production than old mice fed the vitamin E diet or young mice fed either diet. There was no significant age difference in IL‐6, IL‐1β, or tumour necrosis factor‐α (TNF‐α) production by splenocytes. Young mice fed the vitamin E‐supplemented diet had significantly lower IL‐1β (day 7) and TNF‐α production (day 5) compared with those fed the control diet. Old mice fed the vitamin E‐supplemented diet had significantly lower TNF‐α production (day 2) than those fed the control diet. Our results indicate that the vitamin E‐induced decrease in influenza viral titre is mediated through enhancement of Th1 cytokines, which may be the result of reduced PGE2 production caused by vitamin E.
Activation of T‐cell receptor‐γδ+ cells in the intestinal epithelia of KN6 transgenic mice
Authors: Kawaguchi‐Miyashita M. , Shimada S. , Matsuoka Y. , Ohwaki M. , Nanno M.
Published: March 2003

We analysed the properties of intraepithelial lymphocytes of small intestine (SI‐IEL) in KN6‐transgenic (Tg) mice expressing cDNA of T‐cell receptor (TCR)‐γδ specific for the T22b molecule. While most splenic Tg TCR‐γδ+ cells from KN6‐Tg mice with H‐2d/d background (Tgd/d mice) were Thy‐1+ CD8α– CD44dull+ CD45RB+ CD69–, Tg TCR‐γδ+ cells in SI‐IEL (Tg γδ‐IEL) were heterogeneous in the expression of Thy‐1, CD8α and CD44 molecules and predominantly CD45RB+ CD69+. Tg γδ‐IEL exhibited a much reduced proliferative response to the antigen (irradiated H‐2b splenocytes) than splenic Tg TCR‐γδ+ cells; the CD44+ subset, but not the CD44– subset, in Tg γδ‐IEL responded to the antigen. Furthermore, Tg γδ‐IEL, but not splenic Tg TCR‐γδ+ cells, displayed cytolytic activity whether they were prepared from conventional or germ‐free KN6‐Tg mice. Comparative analysis of young and aged KN6‐Tg mice revealed that the proportion of CD44+ cells in Tg γδ‐IEL increased but the proliferative response of Tg γδ‐IEL to the antigen attenuated in association with ageing. Moreover, although Tg γδ‐IEL from Tgb/d mice contained a higher proportion of CD44+ cells than Tgd/d mice, they did not respond to the antigen. These results demonstrate that Tg TCR‐γδ+ cells lose the ability to recognize the antigen following activation in the intestinal epithelia.
Nitric oxide and virus infection
Authors: Akaike T. , Maeda H.
Published: March 2003

Nitric oxide (NO) has complex and diverse functions in physiological and pathophysiological phenomena. The mechanisms of many events induced by NO are now well defined, so that a fundamental understanding of NO biology is almost established. Accumulated evidence suggests that NO and oxygen radicals such as superoxide are key molecules in the pathogenesis of various infectious diseases. NO biosynthesis, particularly through expression of an inducible NO synthase (iNOS), occurs in a variety of microbial infections. Although antimicrobial activity of NO is appreciated for bacteria and protozoa, NO has opposing effects in virus infections such as influenza virus pneumonia and certain other neurotropic virus infections. iNOS produces an excessive amount of NO for long periods, which allows generation of a highly reactive nitrogen oxide species, peroxynitrite, via a radical coupling reaction of NO with superoxide. Thus, peroxynitrite causes oxidative tissue injury through potent oxidation and nitration reactions of various biomolecules. NO also appears to affect a host's immune response, with immunopathological consequences. For example, overproduction of NO in virus infections in mice is reported to suppress type 1 helper T‐cell‐dependent immune responses, leading to type 2 helper T‐cell‐biased immunological host responses. Thus, NO may be a host response modulator rather than a simple antiviral agent. The unique biological properties of NO are further illustrated by our recent data suggesting that viral mutation and evolution may be accelerated by NO‐induced oxidative stress. Here, we discuss these multiple roles of NO in pathogenesis of virus infections as related to both non‐specific inflammatory responses and immunological host reactions modulated by NO during infections in vivo.
Two independent pathways of maternal cell transmission to offspring: through placenta during pregnancy and by breast‐feeding after birth
Authors: Zhou L. , Yoshimura Y. , Huang Y.‐Y. , Suzuki R. , Yokoyama M. , Okabe M. , Shimamura M.
Published: March 2003

Cell transmission from mother to offspring was demonstrated using mice with green fluorescent protein (GFP) transgenic markers. GFP transgene heterozygous (+/−) females were mated with GFP (−/−) males, and GFP+ cells in the GFP (−/−) fetuses generated between them were analysed to assess maternal blood cell transmission to conceptuses in utero. The GFP+ maternal cells were observed throughout the body of the fetuses, as shown by fluorescence stereomicroscopy. Cell entrance into the fetal immune system was shown by histochemical and flow cytometric analyses of fetal organs such as thymus, spleen and liver. The GFP+ maternal cells persisted in the offspring until postpartum. Next, GFP (−/−) neonates fed by GFP+ foster mothers were examined to study the transfer of maternal milk leucocytes to offspring through breast‐feeding. GFP+ leucocytes that had infiltrated through the wall of the digestive tract were mainly localized in the livers of neonates. Their accumulation in the livers reached a maximum on days 5 or 6, and these cells became undetectable, as assessed by either histochemistry or flow cytometry, after day 9 of starting foster nursing. Collectively, the present results demonstrate two independent pathways of maternal cell transmission to offspring: transplacental passage during pregnancy and breast‐feeding after birth.
Studies of delayed systemic effects of ultraviolet B radiation (UVR) on the induction of contact hypersensitivity, 2. Evidence that interleukin‐10 from UVR‐treated epidermis is the critical mediator
Authors: Kurimoto I. , Kitazawa T. , Streilein J. W.
Published: March 2003

Acute, low‐dose ultraviolet B radiation (UVR) alters cutaneous immunity at the local site as well as systemically. Within 2–3 days of UVR exposure, recipient mice lose their capacity to develop contact hypersensitivity (CH) when hapten is painted on unexposed skin. This loss correlates temporally with a functional deficit among dendritic antigen‐presenting cells within non‐draining lymph nodes and spleen. In the experiments described, the delayed systemic immune deficiency following acute, low‐dose UVR exposure was found to be eliminated with neutralizing anti‐interleukin‐10 (IL‐10) antibody. Intracutaneous injection of IL‐10 generated a deficiency of systemic immunity as well as a functional deficit among lymph node dendritic cells that was similar to that induced by UVR. The skin itself was found to be the source of the IL‐10 responsible for these defects, and epidermis (presumably keratinocytes) rather than mast cells was found to be the source of IL‐10 within UVR‐exposed skin. The potential relationships are discussed between the delayed systemic immune deficit created by acute, low‐dose UVR, and the systemic immune deficits caused by chronic, high‐dose UVR and by a single, high‐dose UVR exposure.
Human amniotic fluid lacks interleukin‐2 and interleukin‐15 but can interact with the β‐chain of the interleukin‐2 receptor
Authors: Searle R. F. , Bromage S. J. , Palmer J. , Curry J. E. , Lang A. K.
Published: March 2003

The present study investigated whether an explanation for the conflicting reports on the interleukin‐2 (IL‐2) status of amniotic fluid is due to the presence of IL‐15 which shares biological activities with IL‐2 and utilizes the IL‐2 receptor β‐chain. Amniotic fluids from 45 normally progressing pregnancies between 14 and 16 weeks after the last menstrual period were assayed for IL‐2 and IL‐15 by bioassay and enzyme‐linked immunosorbent assay (ELISA). The ability of amniotic fluids to induce cytotoxic T lymphoblastoid line‐2 (CTLL‐2) cell proliferation was demonstrated to be dependent upon bioassay culture conditions. In serum‐free medium each amniotic fluid stimulated CTLL‐2 proliferation with a mean level of IL‐2‐like bioactivity of 14·7 ± 2·3 ng/ml but amniotic fluids failed to induce CTLL‐2 proliferation in serum‐supplemented medium. Treatment with neutralizing anti‐IL‐2 or anti‐IL‐15 antibodies failed to inhibit amniotic fluid‐induced CTLL cell proliferation in serum‐free medium, indicating a lack of IL‐2 and IL‐15 bioactivity. In contrast, treatment with anti‐IL‐2 receptor β‐chain antibody significantly reduced amniotic fluid‐induced proliferation. The lack of IL‐2 and IL‐15 activity in amniotic fluids was confirmed using ELISA. Although high levels of IL‐15 immunoactivity were detected in all samples, specificity controls showed a lack of specific IL‐15 immunoactivity in amniotic fluid. Pretreatment of amniotic fluids with 100–500 ng/ml mouse immunoglobulin G abrogated IL‐15 immunoactivity, indicating that amniotic fluid contains molecules binding to Fc regions of immunoglobulins and responsible for false ELISA positivity. These studies unequivocally show that amniotic fluid lacks IL‐2 and IL‐15 but can stimulate CTLL‐2 cell proliferation via the IL‐2 receptor β‐chain. The absence of IL‐2 and IL‐15 in normal mid‐trimester amniotic fluid suggests that the cytokine profile of human pregnancy appears to be associated with a bias against type 1 cytokines within the feto–placental unit.
The identification of plant lectins with mucosal adjuvant activity
Authors: Lavelle E. C. , Grant G. , Pusztai A. , Pfüller U. , O'hagan D. T.
Published: March 2003

To date, the most potent mucosal vaccine adjuvants to be identified have been bacterial toxins. The present data demonstrate that the type 2 ribosome‐inactivating protein (type 2 RIP), mistletoe lectin I (ML‐I) is a strong mucosal adjuvant of plant origin. A number of plant lectins were investigated as intranasal (i.n.) coadjuvants for a bystander protein, ovalbumin (OVA). As a positive control, a potent mucosal adjuvant, cholera toxin (CT), was used. Co‐administration of ML‐I or CT with OVA stimulated high titres of OVA‐specific serum immunoglobulin G (IgG) in addition to OVA‐specific IgA in mucosal secretions. CT and ML‐I were also strongly immunogenic, inducing high titres of specific serum IgG and specific IgA at mucosal sites. None of the other plant lectins investigated significantly boosted the response to co‐administered OVA. Immunization with phytohaemagglutinin (PHA) plus OVA elicited a lectin‐specific response but did not stimulate an enhanced response to OVA compared with the antigen alone. Intranasal delivery of tomato lectin (LEA) elicited a strong lectin‐specific systemic and mucosal antibody response but only weakly potentiated the response to co‐delivered OVA. In contrast, administration of wheatgerm agglutinin (WGA) or Ulex europaeus lectin 1 (UEA‐I) with OVA stimulated a serum IgG response to OVA while the lectin‐specific responses (particularly for WGA) were relatively low. Thus, there was not a direct correlation between immunogenicity and adjuvanticity although the strongest adjuvants (CT, ML‐I) were also highly immunogenic.
Interleukin (IL)‐18 induces Langerhans cell migration by a tumour necrosis factor‐α‐ and IL‐1β‐dependent mechanism
Authors: Cumberbatch M. , Dearman R. J. , Antonopoulos C. , Groves R. W. , Kimber I.
Published: March 2003

Following skin sensitization a proportion of epidermal Langerhans cells (LC) are stimulated to leave the skin and to migrate, via afferent lymphatics, to draining lymph nodes where they accumulate as immunostimulatory dendritic cells (DC). It has been demonstrated previously that tumour necrosis factor‐α (TNF‐α), an inducible product of epidermal keratinocytes, and interleukin (IL)‐1β, produced exclusively by LC in murine epidermis, provide important signals for the initiation of this response. Recently, it has been demonstrated that IL‐18, a cytokine produced by both LC and keratinocytes within the epidermis, may also participate in immune responses induced following skin sensitization. In the present investigations, the ability of IL‐18 to contribute to the regulation of LC migration and the accumulation of DC in draining lymph nodes has been examined. It was found that, like IL‐1β, IL‐18 administered intradermally to mice resulted in a significant reduction in epidermal major histocompatibility complex (MHC) class II+ LC densities and a marked increase in lymph node DC numbers. Using neutralizing anti‐TNF‐α and blocking anti‐type I IL‐1 receptor (IL‐1RI) antibodies, it was shown also that the induction by IL‐18 of both LC mobilization and DC accumulation in regional lymph nodes was dependent upon availability of TNF‐α and the integrity of IL‐1RI signalling. Furthermore, using IL‐1β converting enzyme (caspase‐1) knockout mice, IL‐18‐induced LC migration was found to have a mandatory requirement for active IL‐1β. Importantly, not only was IL‐18 able to contribute to the regulation of LC migration, it was found to be essential for the manifestation of these processes in response to topical sensitization with the contact allergen oxazolone.
The 3′‐untranslated region of human interleukin‐8 mRNA suppresses IL‐8 gene expression
Authors: Yu Y. , Chadee K.
Published: March 2003

Although adenosine/uridine (AU)‐rich sequences in the 3′‐untranslated region (UTR) of the interleukin‐8 (IL‐8) gene have been suggested to contribute to its post‐transcriptional regulation, the molecular basis whereby this occurs still needs to be understood. To investigate the role of the 3′‐UTR on human IL‐8 gene regulation, chimeric reporter genes were generated by adding full length or differentially deleted 3′‐UTR of the IL‐8 gene to chloramphenicol acetyltransferase (CAT). Addition of the entire IL‐8 3′‐UTR markedly reduced CAT mRNA and protein expression in COS 7 cells. In a reporter gene study, IL‐8 3′‐UTR destabilized CAT mRNA, which was dependent on active transcription in COS 7 cells. A 357‐base sequence (nucleotides (nt) 2387–2743 of genomic DNA) within 3′‐UTR, designated e, suppressed CAT gene expression by accelerating CAT mRNA turnover. A 26‐base AU‐rich sequence (nt 2552–2577) within e, containing four AUUUA pentamers that form two UAUUUAUU and one UUAUUUAU octamers, did not suppress CAT gene expression. However, deletion of the AU‐rich sequences attenuated the inhibitory effect of e on CAT gene expression. Elimination of the first 100 bases (nt 2386–2486) attenuated the potency of fragment e, but much weaker than elimination of the first 146 bases (nt 2387–2533). This study gives new insights in unravelling the molecular mechanisms involved in the post‐transcriptional regulation of the IL‐8 gene.
CD8+ T cells in human uterine endometrial lymphoid aggregates: evidence for accumulation of cells by trafficking
Authors: Yeaman G. R. , Collins J. E. , Fanger M. W. , Wira C. R.
Published: March 2003

Lymphoid aggregates (LA) develop during the proliferative phase of the menstrual cycle in the human uterine endometrium (EM). They contain mostly CD8+ T cells and B cells. As these LA are absent immediately following menses, they may arise by division of cells resident in the EM, or by division of a limited number of precursor cells that traffic into the EM during the early proliferative phase of the menstrual cycle. Alternatively, they may arise by the continuous trafficking of cells into the EM throughout the proliferative phase of the menstrual cycle. In this study we investigated the distribution and frequency of CD8+ T cells in the aggregates using expression of Vβ2 or Vβ8 as markers of clonality and Ki‐67 as a marker of dividing cells. Confocal microscopic analysis of endometrial tissues showed the random distribution of CD8+ T cells within aggregates within the same sample and in aggregates from different samples. Furthermore, comparisons of the distribution of Vβ2 and Vb8 with expected values predicted from Poisson distribution values were not significantly different, suggesting that CD8+ T cells do not arise by division from single precursors. A low level of T‐cell division within LAs was confirmed by positive staining for Ki‐67. Dividing T cells were randomly dispersed throughout the LA and the frequency of dividing cells did not vary greatly between aggregates within the same tissue. Nearest‐neighbour analysis of dividing cells showed no statistically significant deviations from a random distribution. Taken together, these results suggest that LA develop during the menstrual cycle largely by the trafficking of cells to nucleation sites within the EM, rather than by division of a limited number of precursor cells.
Mycobacterium avium infection in CD14‐deficient mice fails to substantiate a significant role for CD14 in antimycobacterial protection or granulomatous inflammation
Authors: Ehlers Stefan , Reiling Norbert , Gangloff Sophie , Woltmann Alexander , Goyert Sanna
Published: March 2003

CD14 is a pattern‐recognition receptor implicated in the inflammatory response to microbial components such as lipopolysaccharide, peptidoglycan and lipoarabinomannan. In this work, we made use of CD14‐deficient (CD14−/−) mice to evaluate the relative importance of CD14 in response to infection with viable, intact cells of Mycobacterium avium in vitro and in vivo. Following co‐incubation of either bone marrow‐derived macrophages (Mφ) or thioglycollate‐elicited peritoneal Mφ from CD14−/− mice with viable M. avium, tumour necrosis factor (TNF) production was significantly reduced and delayed compared to TNF secretion by infected CD14+/+ Mφ. However, following intravenous infection with a M. avium strain of either high virulence (TMC724) or intermediate virulence (SE01), there was no difference in the bacterial loads of lungs, livers or spleens at 3, 5 and 8 weeks postinfection in CD14−/− mice when compared with syngeneic CD14+/+ mice. At these time‐points, TNF and interferon‐γ (IFN‐γ) mRNA expression in the liver was similar in infected CD14+/+ and CD14−/− mice, and granuloma formation and expression of inducible nitric oxide synthase within granuloma Mφ was the same in both mouse groups. In conclusion, although the absence of CD14 results in significantly reduced and delayed TNF production in response to stimulation with M. avium in vitro, there is no evidence that CD14 plays a significant role in either the antibacterial defence or the chronic granulomatous reaction to M. avium infection in vivo.
Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes
Authors: Fernie‐King Barbara A. , Seilly David J. , Willers Christine , Würzner Reinhard , Davies Alexandra , Lachmann Peter J.
Published: March 2003

Streptococcal inhibitor of complement (SIC) was first described in 1996 as a putative inhibitor of the membrane attack complex of complement (MAC). SIC is a 31 000 MW protein secreted in large quantities by the virulent Streptococcus pyogenes strains M1 and M57, and is encoded by a gene which is extremely variable. In order to study further the interactions of SIC with the MAC, we have made a recombinant form of SIC (rSIC) in Escherichia coli and purified native M1 SIC which was used to raise a polyclonal antibody. SIC prevented reactive lysis of guinea pig erythrocytes by the MAC at a stage prior to C5b67 complexes binding to cell membranes, presumably by blocking the transiently expressed membrane insertion site on C7. The ability of SIC and clusterin (another putative fluid phase complement inhibitor) to inhibit complement lysis was compared, and found to be equally efficient. In parallel, by enzyme‐linked immunosorbent assay both SIC and rSIC bound strongly to C5b67 and C5b678 complexes and to a lesser extent C5b‐9, but only weakly to individual complement components. The implications of these data for virulence of SIC‐positive streptococci are discussed, in light of the fact that Gram‐positive organisms are already protected against complement lysis by the presence of their peptidoglycan cell walls. We speculate that MAC inhibition may not be the sole function of SIC.
The role of prostaglandin E2 in the immunopathogenesis of experimental pulmonary tuberculosis
Authors: Rangel Moreno Javier , Estrada García Iris , De La Luz García Hernández María , Aguilar Leon Diana , Marquez Ricardo , Hernández Pando Rogelio
Published: March 2003

Prostaglandins (PG) are potent mediators of intercellular communication, and PGE2 at high concentration is immunosuppressive for T‐cell‐mediated immunity. We studied the kinetics of PGE2 production and the expression of the enzymes related to its synthesis during the course of experimental pulmonary tuberculosis. Secondly, we analysed the pathological and immunological changes produced by the pharmacological suppression of PG production. In BALB/c mice infected via the trachea with Mycobacterium tuberculosis H37Rv there is an initial phase of partial resistance, dominated by type 1 cytokines plus tumour necrosis factor‐α (TNF‐α) and expression of the inducible form of nitric oxide synthase (iNOS), followed by a phase of progressive disease. During the early phase of the infection some activated macrophages located in the alveolar‐capillary interstitium and in granulomas showed strong PGE2 immunostaining. However, PGE2 concentrations were relatively low and stable. Animals in this early phase of infection were treated with niflumic acid, a potent and specific blocker of cyclo‐oxygenase 2, the rate‐limiting enzyme of PG production. In comparison with control animals, the suppression of PG synthesis produced higher inflammation and expression of TNF‐α, interleukin‐1α and interferon‐γ (IFN‐γ), but almost complete disappearance of iNOS expression, which coexisted with a significant increment of bacterial load. The late progressive phase in this experimental model is characterized by progressive pneumonia, small granulomas and diminished expression of IFN‐γ, TNF‐α and iNOS in coexistence with high expression of IL‐4. Strong PGE2 immunostaining was seen in foamy macrophages localized in the pneumonic areas, and the PGE2 concentration was four‐fold higher in this late phase of infection than during the early phase. When PG production was suppressed in animals suffering advanced phase infection, a significant reduction of pneumonia and bacillus load with striking increment of granuloma size was seen, and the expression of IFN‐γ, TNF‐α and iNOS was also improved. These findings demonstrate a significant participation of PGE2 in the pathogenesis of pulmonary tuberculosis, showing that during the early phase of the infection there are low PGE2 concentrations which contribute to iNOS expression permitting the temporal control of bacillus growth, while the high PGE2 concentrations during the late phase of the disease contribute to down‐regulate cell‐mediated immunity, permitting disease progression.