The modulation of expression of CD80 and CD86 on T cells following infection with human T lymphotropic virus (HTLV)-I/II and its functional importance in T-T cell interactions was examined. Infection with HTLV-I/II leads to constitutive expression of CD80 and CD86, concomitant to down-modulation of CD28 on T cells. The CD80/CD86+ HTLV-infected T cells stimulated proliferation of allogeneic and autologous resting T cells, which could be specifically blocked by a soluble CTLA-4Ig chimeric protein, anti-CD80 or anti-CD86, but not by anti-CD54. It was necessary to inhibit interaction with both ligands (CD80 and CD86) to optimally block HTLV-mediated proliferation of allogeneic and autologous resting T cells. Simultaneous addition of anti-CD8O and anti-CD86 Abs also inhibited production of IFN-gamma, TNF-alpha, and IL-4, with no effect on IL-10 production, for both allo- and autologous T cell proliferation. Further, there was a direct correlation between the spontaneous proliferation of lymphocytes from patients infected with HTLV-II and expression of CD80, which could be blocked by simultaneous addition of anti-CD80 and anti-CD86. Taken together, these results suggest that HTLV-infected CD80/CD86+ T cells serve as APCs, leading to a sustained proliferation of T cells, and that both ligands participate in allostimulation, autologous proliferation, as well as spontaneous proliferation of HTLV-II-infected PBMC.

The aim of our study was to identify differences in cell surface marker expression between T cells taken from the peripheral blood (PB) of healthy individuals and T cells recovered from inflamed joints of rheumatoid arthritis (RA) patients. Out of 118 monoclonal antibodies (MoAbs) directed against activation antigens on haematopoietic cells, 12 MoAbs recognizing nine distinct surface molecules were selected after a screening procedure to study the expression of the corresponding antigens on T cells from the PB, synovial fluid and synovial tissue of RA patients, and also on T cells from PB and spleens of controls. Using two-colour flow cytometry and immunohistology we found the molecules B-C5, CD39, CD40, CD45 R0, CD54, CD76 and potentially 1D11 to be substantially up-regulated on T cells from various body compartments in RA patients. We thus could determine that the cell surface of T cells in RA patients not only differs in MHC class II expression, but also in a number of other activation-associated cell surface molecules from T cells in healthy individuals.

To further investigate the immunomodulatory effects of dietary lipids, rats were fed on a low-fat diet or on high-fat diets that contained hydrogenated coconut, olive, safflower, evening primrose or fish oil as the principal fat source. The fish oil diet decreased the level of expression of CD2, CD11a, CD18 and CD44 on the surface of freshly prepared lymphocytes and of CD2, CD11a, CD18, CD54 (intercellular adhesion molecule-1; ICAM-1) and CD62L (L-selectin) on the surface of concanavalin A (Con A)-stimulated lymphocytes. The olive oil diet also resulted in decreased expression of some adhesion molecules. The fish or olive oil diets, and to a lesser extent the safflower or evening primrose oil diets, decreased the adhesion of both freshly prepared and Con A-stimulated lymphocytes to macrophage monolayers. The fish oil diet, and to a lesser extent the olive or evening primrose oil diets, reduced the ability of Con A-stimulated lymphocytes to adhere to untreated endothelial cells. Furthermore, the fish oil diet resulted in a 50% reduction in Con A-stimulated lymphocyte adhesion to tumour necrosis factor-alpha (TNF-alpha)-stimulated endothelial cells. This study demonstrates that dietary lipids affect the expression of functionally important adhesion molecules on the surface of lymphocytes. Furthermore, this study suggests that such diet-induced effects on adhesion molecule expression might alter the ability of lymphocytes to bind to macrophages and to endothelial cells. Of the diets studied fish oil causes the most significant effects. The results of this study suggest that a reduction in cellular infiltration may partly explain the protective effect of a fish-oil-rich diet against the development of inflammatory and cardiovascular diseases.

Although fumaric acid esters (FAE) have a decade-long firm place in the therapeutic armamentarium for psoriasis, their pleiotropic mode of action is not yet fully understood. While most previous studies have focused on the effects of FAE on leucocytes, we have addressed their activity on macro- and microvascular endothelial cells. As detected both on mRNA and protein levels, dimethylfumarate effected a profound reduction of TNFα-induced expression of E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106) on two different endothelial cell populations in a concentration-dependent manner. This reduction of several endothelial adhesion molecules was accompanied by a dramatic diminution of both rolling and firm adhesive interactions between endothelial cells and lymphocytes in a dynamic flow chamber system. Dimethylfumarate, at a concentration of 50 μm, reduced lymphocyte rolling on endothelial cells by 85.9% (P<0.001 compared to untreated controls), and it diminished the number of adherent cells by 88% (P<0.001). In contrast, monomethylfumarate (MMF) influenced neither surface expression of adhesion molecules nor interactions between endothelial cells and lymphocytes. These observations demonstrate that endothelial cells, in addition to the known effects on leucocytes, undergo profound functional changes in response to dimethylfumarate. These changes are accompanied by severely impaired dynamic interactions with lymphocytes, which constitute the critical initial step of leucocyte recruitment to inflamed tissues in psoriasis and other TNF-related inflammatory disorders.

Immunosuppressive effects of low levels of ultraviolet B (UVB) radiation on cutaneous immune responses have been attributed to deleterious effects of UVB radiation on epidermal Langerhans cells (LC). To determine how UVB radiation modulates LC function we examined the effect of in vitro UVB exposure on LC accessory cell activity and surface phenotype. Exposure of BALB/c murine epidermal cells to low dose (less than or equal to 200 J/m2) UVB radiation in vitro inhibited their ability to support the mitogenic response of unstimulated, accessory cell-depleted splenic T cells to anti-CD3 mAb. LC accessory cell activity was also inhibited when LC were exposed to UVB radiation in situ, although several-fold higher doses of UVB radiation were required to achieve complete inhibition of LC function. This dose-dependent inhibition was mediated through a direct effect on LC that could not be reversed by IL-1 or IL-6 alone or in combination, or granulocyte-macrophage-CSF. TNF-alpha did not inhibit LC accessory cell function in this assay and anti-TNF-alpha neutralizing antibodies did not reverse the inhibitory effects of UVB radiation. UVB irradiated LC failed to participate in the anti-CD3-dependent clustering that normally occurs between T cells and LC during the proliferative response of murine T cells to anti-CD3 mAb, suggesting that UV radiation may interfere with accessory cell function by preventing intercellular adhesion. Two-color flow cytometric studies revealed low levels of the ICAM-1 on freshly isolated LC and some keratinocytes. ICAM-1 expression on LC increased 15 to 20-fold within the first 24 h in vitro and continued to increase during a 72-h culture period. The integrin LFA-1 was not identified on freshly isolated or cultured LC but was detected on responding T cells. Prior exposure of LC to UVB radiation (50 or 100 J/m2) inhibited the increase in ICAM-1 expression that normally occurs in vitro by up to 70% whereas surface levels of class II MHC Ag, CD45 and Fc-gamma receptors were not affected. Blocking studies revealed that anti-CD3 induced T cell proliferation and T cell-LC cluster formation was inhibited by both anti-LFA-1 and anti-ICAM-1 mAb suggesting that ICAM-1 expressed on LC must bind to LFA-1 on T cells to facilitate proliferative responses of T cells to anti-CD3 mAb. We conclude that the in vitro inhibitory effects of low dose UVB radiation on LC accessory function may result because UVB radiation prevents upregulation of ICAM-1 expression by LC in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

In addition to the interaction between the TCR and the MHC/Ag complex on the APC, optimal T cell activation also requires interaction between adhesion molecules on the APC and their ligands on T cells. We determined the presence of adhesion molecules on human epidermal Langerhans cells (LC) and their role in Ag-specific T cell activation. Freshly isolated LC did not display ICAM-1 (CD54), ICAM-2, LFA-1 (CD11a), and LFA-3 (CD58), as detected by double-color FACS analysis, using HLA-DR expression for LC identification. Upon culture, LC clearly expressed ICAM-1 and LFA-3, both already detectable after 1 day, reaching a plateau at day 2. ICAM-2 and LFA-1 were undetectable on cultured LC and attempts to induce this expression by different culture conditions remained unsuccessful. mAb against ICAM-1, LFA-1, LFA-3, and CD2, continuously present during culture, inhibited the T cell proliferative response to Candida albicans presented by cultured LC. Pretreatment of LC and/or T cells with mAb indicated that anti-ICAM-1 and anti-LFA-3 inhibited at the LC level, whereas anti-LFA-1 and anti-CD2 inhibited at the T cell level. The mAb-induced inhibition was dose-dependent, but a total blockade of the response was never achieved. Time-course observations revealed that ICAM-1 and LFA-3 on LC only functioned during the initiation phase of T cell activation. Our study demonstrates that both ICAM-1 and LFA-3 on LC considerably contribute to the generation of a T cell response. The high expression of these accessory molecules enable LC, at least in part, to perform their powerful Ag-presenting function.

The existence of endothelial progenitor cells (EPC) with high cell-cycle rate in human umbilical cord blood has been recently shown and represents a challenging strategy for therapeutic neovascularization. To enhance knowledge for future cellular therapy, we compared the phenotypic, functional and gene expression differences between EPC-derived cells generated from cord blood CD34(+) cells, and lymphatic and macrovascular endothelial cells (EC) isolated from human foreskins and umbilical veins, respectively. Under appropriate culture conditions, EPC developed into fully matured EC with expression of similar endothelial markers as lymphatic and macrovascular EC, including CD31, CD36, von Willebrand factor FVIII, CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-cadherin), Tie-1, Tie-2, VEGFR-1/Flt-1 and VEGFR-2/Flk-1. Few EPC-derived cells became positive for LYVE-1, indicating their origin from haematopoietic stem cells. However they lacked expression of other lymphatic cell-specific markers such as podoplanin and Prox-1. Functional tests demonstrated that the cobblestone EPC-derived cells up-regulated CD54 and CD62E expression in response to TNF-alpha, incorporated DiI-acetylated low-density liproprotein and formed cord- and tubular-like structures with capillary lumen in three-dimensional collagen culture--all characteristic features of the vascular endothelium. Structures compatible with Weibel-Palade bodies were also found by electron microscopy. Gene microarray profiling revealed that only a small percentage of genes investigated showed differential expression in EPC-derived cells and lymphatic EC. Among them were adhesion molecules, extracellular matrix proteins and cytokines. Our data point to the close lineage relationship of both types of vascular cells and support the theory of a venous origin of the lymphatic system.

BACKGROUND

Dendritic cells (DCs) can take up an array of different antigens, including microorganisms which they can process and present more effectively than any other antigen presenting cell. However, whether the interaction between the human DC and Mycobacterium tuberculosis represents a defense mechanism by the invaded host, or helping the invader to evade the defense mechanism of the host is still not clearly understood.

RESULTS

To analyze the interactions between M. tuberculosis and immune cells, human peripheral blood monocyte-derived immature DCs were infected with M. tuberculosis H37Rv wild type strain and flow cytometry was used to analyse cell surface expression markers. The ability of the M. tuberculosis infected DC to induce T cell proliferation using 5 and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution technique was also investigated. DCs were found to internalize the mycobacteria and show dose dependent infection and necrosis with different multiplicity of infection. Flow cytometry analysis of cell surface expression markers CD40, CD54, CD80, CD83, CD86 and HLA DR in infected DC revealed significant (p < 0.05) up regulation following infection with M. tuberculosis in comparison to immature DC with no stimulation. Lipopolysaccharide (LPS) from Salmonella abortus equi, a known DC maturation agent, was used as a positive control and showed a comparable up regulation of cell surface markers as observed with M. tuberculosis infected DC. It was revealed that the M. tuberculosis infected DC induced T cell proliferation.

CONCLUSIONS

These data clearly demonstrate that M. tuberculosis induces activation and maturation of human monocyte-derived immature DC as well as induces T cell proliferation in vitro.

Cytomegalovirus (CMV) is a major pathogen in transplant recipients and AIDS patients, and the virus may also play a role in allograft rejection. Previous work from this laboratory demonstrated increased cell surface expression of the adhesion molecules ICAM-1 (CD54) and LFA-3 (CD58) following CMV infection in vitro. We investigated whether the induction of adhesion molecules by CMV was a direct viral effect or secondary to cytokine induction. Cytokines known to up-regulate ICAM-1, such as TNFalpha or IL-1beta, were not detected in the supernatants of infected fibroblasts, and neutralizing antibodies against these cytokines did not abrogate the induction of either ICAM-1 or LFA-3 by CMV. Infected cell supernatants had increased levels of IL-6, IL-8 and IFNbeta however, the addition of recombinant forms of these cytokines did not affect adhesion molecule expression. Neither virus-free infected cell supernatants nor UV-inactivated virus up-regulated adhesion molecules, demonstrating that the induction of ICAM-1 and LFA-3 by CMV was a direct effect requiring infectious virus. Effective antiviral treatment with ganciclovir or foscarnet accentuated rather than abrogated the up-regulation of adhesion molecules, suggesting that CMV immediate early/early gene expression, which is not blocked by such treatment, was responsible for the adhesion molecule induction. Thus, despite effective antiviral therapy in the transplant recipient, CMV infected cells may continue to provide a focus of proinflammatory activity, which could contribute to immunopathology and/or accentuate graft rejection or graft-versus-host disease in vivo.

We studied the effect of monoclonal antibodies (mAbs) against CD54 (intercellular adhesion molecule-1; ICAM-1) and CD11a (lymphocyte function-associated antigen-1; LFA-1) on the prevention and treatment of endotoxin-induced uveitis (EIU). When treated at the time of endotoxin injection the mean number of inflammatory cells infiltrating the eye +/- S.E.M. on histologic sections was 469.2 +/- 51.9 for controls, 13.8 +/- 2.6 for rats receiving anti-ICAM-1 mAb (P < 0.0001), and 195.8 +/- 48.8 for rats receiving anti-LFA-1 mAb (P = 0.0003). When treated after the start of inflammatory disease, the mean number of infiltrating inflammatory cells +/- S.E.M. was 273.0 +/- 30.7 for controls, 6.4 +/- 1.7 for rats receiving anti-ICAM-1 mAb (P < 0.0001), and 54.2 +/- 7.6 for rats receiving anti-LFA-1 mAb (P < 0.0001). The mean number of cells per milliliter of aqueous humor +/- S.E.M. was 1867.6 +/- 321.8 for controls, 21.7 +/- 5.3 for rats receiving anti-ICAM-1 mAb (P < 0.0001), and 295.1 +/- 71.2 for rats receiving anti-LFA-1 mAb (P < 0.0001). MAbs against ICAM-1 and LFA-1 significantly inhibited the development of EIU and were effective in treating clinically evident ocular inflammatory disease.

We performed a cross-sectional flow cytometric analysis of peripheral blood mononuclear cells to evaluate human immunologic status during early stages of Trypanosoma cruzi infection in children. We identified major immunological features corresponding to three proposed phases of disease: early acute (EA) phase, late acute (LA) phase and recent chronic (RC) phase. EA phase was accompanied by expansion of conventional B cells, up-regulation of CD54 on monocytes and down-regulation of CD54 on T cells and not associated with monocyte-activation phenotypes or changes of natural killer (NK) population. LA phase was characterized by a selective increase in a distinct lineage of NK cells (CD16+CD56-), as well as a persistent expansion of B cells and down-regulation of CD54 on T cells. RC phase showed persistent low levels of CD54 molecule on T cells and an increase of B cells, mainly triggered by expansion of the B1-cell subset, as well as increased expression of human leucocyte antigen (HLA-DR) by monocytes. These findings reinforce the hypothesis that T. cruzi-derived antigens are able to activate NK cells before the development of T-cell-mediated immunity. Moreover, our data support previous findings of increased levels of B1 lymphocytes during human Chagas' disease and show that this event is already present during initial stages of chronic infection.

Eosinophils from sputum, nasal polyps, and bronchoalveolar lavages of asthmatics demonstrated a considerably increased CD11b expression, compared with blood eosinophils. Furthermore, the tissue eosinophils expressed ICAM-1 (CD54) and HLA-DR, whereas peripheral blood eosinophils did not. In vitro migration of peripheral blood eosinophils across IL-1-activated human umbilical vein endothelial cell monolayers caused a considerable up-regulation of CD11b and CD35 expression, no induction of ICAM-1 or HLA-DR, and a small but significant decrease in CD11a, CD29, and CD32 expression. These changes were only partially inducible with supernatants from nonactivated or IL-1-activated endothelial cells, platelet-activating factor, or a variety of recombinant cytokines. Thus, cell-cell interactions mediated by receptor-ligand binding or endothelial cell membrane-bound mediators, rather than soluble factors, are responsible for the altered eosinophil surface marker expression. Indeed, preparations of membrane fragments from IL-1-stimulated endothelial cells were able to induce up-regulation of CD11b, which was not inhibitable with the platelet-activating factor antagonist WEB 2086 or antibodies against ELAM-1, VCAM-1, or ICAM-1. Investigation of the functional significance of the increased CD11b expression on eosinophils revealed only minimal changes in the adherence or transmigration capacity. Nevertheless, increased CD11b expression was related to an increased capacity to generate superoxide after stimulation with opsonized zymosan. Thus, cell-cell interactions between eosinophils and endothelial cells induce a considerable up-regulation of CD11b and CD35 on eosinophils and an increased capacity to generate an oxidative burst.

To further understand the mechanism of lymphocyte accumulation within the thyroid gland in autoimmune thyroid disease we have examined the expression, regulation, and functional significance of the intercellular adhesion molecule-1 (ICAM-1, CD54) in human thyroid monolayer cells and immortalized thyroid cell clones. Human thyroid monolayer cells derived from both normal and abnormal human thyroid tissue showed low basal expression of the ICAM-1 antigen by flow cytometric assessment (mean % +/- SD positive cells = 13.7 +/- 6.1) compatible with the presence of ICAM-1 positive nonthyroid cells within the monolayer cultures. However, thyroid cell ICAM-1 antigen expression was further induced by exposure to recombinant human interferon-gamma (IF-gamma). At 100 U/ml, IF-gamma induced ICAM-1 expression in 56.0 +/- 19.0% of thyroid monolayer cells. Even greater expression of ICAM-1 antigen was induced by IF-gamma in human fetal thyroid cell monolayers of high purity (up to 80% of ICAM-1 positivity) thyroid monolayers established from a patient with Graves' disease (up to 84%), and in two immortalized human thyroid cell clones, 12S and TAD-2 (up to 61%). Furthermore, dose-response curves for ICAM-1 and HLA-DR antigen induction by increasing concentrations of IF-gamma showed that ICAM-1 antigen gene induction was 10-fold more responsive to IF-gamma than the HLA-DR antigen gene. In order to explore the functional consequence of ICAM-1 antigen expression by thyroid epithelial cells we examined the binding of peripheral blood mononuclear cells to thyroid monolayer cells and immortalized thyroid cells. These studies revealed a preferential adhesion of human PBMC to IF-gamma-treated thyroid monolayers compared to untreated control monolayer cells. Furthermore, this IF-gamma-induced cell adhesion was specifically inhibited by monoclonal anti-ICAM-1. These experiments demonstrate not only the capacity of human thyroid epithelial cells to express ICAM-1 antigen in the presence of a cytokine but, in addition, identify ICAM-1 antigen as responsible for enhanced T cell binding to thyroid epithelial cells. ICAM-1 antigen may, therefore, play an important role in T cell targeting and accumulation within the thyroid gland in autoimmune thyroid disease.

BACKGROUND

Positive test reactions to epicutaneous application of aeroallergens served as a model of 'early' eczema in atopic dermatitis (AD) in a number of dermato-immunological studies. However, no quantitative evaluation has been performed so far comparing specific T-cell activation parameters in the peripheral blood of AD patients with positive or negative atopy patch tests (APT).

OBJECTIVE

The purpose of this study was to investigate specific immunological parameters in patients with atopic dermatitis showing positive or negative atopy patch tests reactions.

METHODS

APT results (n = 96) were compared with allergen-specific IgE, specific lymphocyte proliferation, and the expression of 'activation' markers on peripheral blood T-cells upon in vitro stimulation with house dust mite, cat or grass pollen allergens.

RESULTS

Only a subpopulation (48%) of patients sensitized to aeroallergens (i.e. specific IgE>> 0.7 kU/L) developed APT-reactions to the corresponding allergen. APT reactions were, however, significantly associated with allergen specific lymphocyte proliferation (p < 0.0001), and a higher number of CD54+ or CD30+ T-cells (p < 0.05) upon in vitro stimulation.

CONCLUSIONS

The association of delayed skin reactions with allergen specific T-cell parameters in the blood points to an immunologically mediated mechanism leading to positive reactions in the APT.

SJL mice provide an interesting paradigm to examine the role(s) of APC in the differential induction of Th1 and Th2 cells. Immunization of young male SJL mice results in the preferential induction of Th2 cells, whereas Th1 cells are induced in age-matched female or older male SJL mice. The absence of Th1 responses in young male mice is associated with in vivo IL-4 and IL-10 down-regulating Mac-3+ APC priming of Th1 cells. The present report examines the mechanism of this APC-dependent induction of Th subsets. Examination of the surface expression of MHC class II, adhesion molecules (CD11a, CD11b, CD48, CD54, and CD102) or costimulatory molecules (CD24, CD80, and CD86) showed no differences between male- and female-derived Mac-3+ APC populations. In addition, no differences were detected in IL-1alpha, IL-1beta, IL-18, TNF-alpha, or IL-12 p35 mRNA expression. However, reduced expression of both IL-10 and IL-12 p40 mRNA were found in Mac-3+ cells from male mice compared with those in Mac-3+ cells from female mice. Anti-IL-4 or anti-IL-10 mAb treatment of young male donor mice eliminated the reduction of both IL-10 and IL-12 p40 mRNA, suggesting that the Th2 inducer phenotype is related to a decreased IL-12 secretion. Consistent with this idea, fewer IL-12 p40-secreting Mac-3+ cells were found in male mice compared with female mice, and treatment with rIL-12 resulted in the priming of Th1 cells in male mice. These data suggest that increased Th2 cytokines in vivo before encounter with Ag inhibit APC expression of IL-12, resulting in the preferential induction of Th2 cells in male SJL mice.

OBJECTIVE

The aim of this study was to prospectively investigate metastatic pathways of spread to lymph node versus bone marrow and identify biological characteristics that determine these patterns in early invasive breast cancer.

METHODS

In all, 177 patients with early invasive breast cancer underwent surgical extirpation of the primary tumour with sentinel lymph node biopsy (SLNB). Bone marrow (BM) aspiration was performed to screen for cytokeratin-positive cells by immunocytochemistry. Lymphatic spread was assessed by histopathological examination of lymph nodes (LN). A representative subset of 87 tumours was analysed by tissue microarray (TMA) to evaluate expression of markers that potentially influence haematogenous vs. lymphatic spread. Patients were followed up for a median of 54.7 months.

RESULTS

Of the 177 patients, 114 (64%) were BM-/LN-, 38 (22%) BM-/LN+, 19 (11%) BM+/LN- and 6 (3%) BM+/LN+. Multivariate analysis of histopathological characteristics revealed that increasing tumour size was significantly associated with both LN positivity (p = 0.003) and BM positivity (p = 0.01), the presence of lymphovascular invasion significantly correlated with LN+ (p = 0.01), whereas lower histological grade was significantly associated with BM+ (p = 0.03). LN+ and BM+ were non-significantly negatively related to each other. Univariate analysis of the TMA data showed differential expression patterns for several factors; significant differences between effects on the two metastatic pathways (lymphatic vs. haematogenous) were found for expression of CD54 (p = 0.03), osteopontin (p = 0.04), bone sialoprotein (p = 0.04) and CXCR4 (p = 0.009). High expression of CD54, osteopontin and bone sialoprotein (BSP) was positively associated with BM + but was either not associated, or negatively associated, with LN+. High CXCR4 expression was positively associated with LN+ and negatively with BM+. High VEGF-C expression was associated with both LN+ and BM+, although this did not attain statistical significance. Due to the small number of clinical events during clinical follow-up, no associations were identified between metastatic spread patterns, recurrence and/or death.

CONCLUSIONS

These findings suggest that distinct lymphatic and haematogenous metastatic pathways exist in early breast cancer and that these pathways are governed by specific biological markers.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) suppresses adaptive immune responses and modulates the function of numerous cells involved in these responses. Our laboratory has shown that dendritic cells (DCs), which are important for the initiation of T-cell-dependent immunity, from TCDD-exposed mice exhibited reduced cell numbers and had altered expression of costimulatory molecules that are critical for the activation of T-cells. To further characterize the effects of TCDD on DCs and to elucidate a potential mechanism of toxicity, we investigated the direct effects of TCDD on DC maturation and survival in vitro. DCs, derived from bone marrow cells, were exposed to TCDD and then treated with TNFalpha to induced maturation. Apoptosis of bone marrow derived DCs (bmDCs) was induced by activating CD95 on the surface of the cells and was measured by annexin V staining. The TCDD-mediated changes in the expression of genes associated with apoptosis were examined using a pathway-specific c-DNA microarray. The results demonstrate that TCDD-treatment of bmDCs enhanced TNFalpha-induced maturation, measured as an increase in the expression of major histocompatibility complex class II, CD86, CD40, and CD54. In addition, TCDD exposure significantly augmented CD95-mediated death of bmDCs and altered the transcription of several genes involved in apoptosis. These findings confirm and extend the in vivo effects of TCDD on DC activation, and suggest that TCDD induces these changes, at least in part, via direct effects on the DC.

The glycoprotein CD86 expressed on APCs provides a costimulatory signal necessary for an efficient activation of naive T cells. In contrast, there is controversy about the condition of expression and the function of CD86 on T cells. In this study, we have analyzed the phenotype and the biological activity of CD86+ T cells generated from human PBMC. Results show that CD86 expression on T cells is induced by long term stimulation via CD3 and IL-2R and is down-regulated as the cells become quiescent. The CD86-expressing cells are memory effector T cells: 1) they express CD45RO and high levels of the activation markers CD25, CD54, and HLA-Dr; 2) they selectively express CD30, CD40-ligand, and CD70; and 3) in response to stimulation, most of them produce IFN-gamma before dying by apoptosis. We then analyzed whether CD86 expressed on T cells is functional. Results show that paraformaldehyde-fixed CD86+ T cells enhance the proliferation and production of IFN-gamma by anti-CD3 mAb-stimulated naive T cells and induce proliferation of resting allogenic T cells. All these effects are prevented by neutralizing anti-CD86 mAbs. In contrast, we report no autocrine effect of CD86 in CD86+ T cell activation. In conclusion, these data show that human memory effector T cells express a functional form of CD86 that can costimulate naive T cell responses.

Associations between hematopoietic cells and their microenvironment are central to the development and maintenance of a functional hematopoietic system. It is important, therefore, to identify the surface receptors that mediate adhesive interactions among hematopoietic cells, stromal cells, and extracellular matrix components. In this study, we examined the expression of mRNA transcripts encoding a number of cell adhesion molecules and surface antigens in primitive hematopoietic cells isolated from murine bone marrow and fetal liver. Using a panel of probes, we hybridized a library of globally amplified cDNA prepared by reverse transcriptase-polymerase chain reaction of poly(A)+ mRNA from individual precursors and mature cell populations, representing precisely defined positions within the hematopoietic developmental hierarchy. The panel included probes specific for the CD45, CD34, P-glycoprotein (mdr1), Ly-6A/E (Sca-1), heat stable antigen (CD24), Fc receptor for IgG FcgammaRII (CD32), CD44, CD22, and ICAM-1 (CD54) genes, as well as alphaL (CD11a), alphaM (CD11b), beta2 (CD18), alpha4 (CD49d), alpha5 (CD49e), beta1 (CD29), and beta7 integrin subunit sequences. The data, which revealed stage- and lineage-specific expression patterns, should prove useful in designing future mechanistic studies aimed at elucidating the role played by adhesion receptors in normal and abnormal hematopoiesis.

The adherence of monocytes to activated endothelium is an early event in atherogenesis. Because antioxidants have been considered to be of antiatherosclerotic potential, we investigated the effects of alpha-tocopherol (TCP) and its acetate and succinate esters on monocyte adhesion to cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Endothelial cells were treated with TCP, alpha-tocopherol acetate (TCP acetate), or alpha-tocopheryl succinate (TCP succinate) before stimulation with tumor necrosis factor-alpha (TNF-alpha; 10 U/ml, 6 h) or interleukin-1 beta (IL-1 beta; 10 U/ml, 6 h). Cytokine-stimulated cell surface expression of vascular cell adhesion molecule-1 (VCAM-1, CD106) and E-selectin (ELAM-1, CD62E), but not of intercellular adhesion molecule-1 (ICAM-1, CD54), was time- and dose-dependently inhibited by TCP succinate but not by TCP or TCP acetate. TCP succinate (200 microM, 24 h) reduced TNF-induced VCAM-1 and E-selectin expression from a specific mean fluorescence intensity of 151 +/- 28 to 12 +/- 4 channels and from 225 +/- 38 to 79 +/- 21 channels, respectively. Succinate alone had no effect. Decreased adhesion molecule expression was associated with a reduction of monocytic cell adhesion. TCP succinate (20 microM, 72 h), but not TCP (200 microM, 72 h), reduced U-937 cell adhesion to TNF-alpha-stimulated (10 U/ml, 6 h) HUVEC by 30% (P < 0.025) and to IL-1 beta-stimulated HUVEC by 56% (P < 0.010). Electrophoretic mobility-shift assays of HUVEC nuclear proteins revealed a decrease in TNF-alpha-stimulated nuclear factor-kappa B (NF-kappa B) activation after pretreatment of HUVEC with TCP succinate but not with TCP, TCP acetate, or succinate alone. In conclusion, we demonstrate that the vitamin E derivative TCP succinate prevents monocytic cell adhesion to cytokine-stimulated endothelial cells by inhibiting the activation of NF-kappa B, further emphasizing the antiatherosclerotic potential of lipid soluble antioxidants.

We have studied the effect of mycophenolate mofetil (MMF), a new drug used in prevention of transplant rejection, on differentiation, maturation and allostimulatory activity of human monocyte-derived dendritic cells (MDDC). MDDC were generated in vitro with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 in the presence or absence of MMF. MMF reduced the number of immature MDDC in culture, dose-dependently, by inducing apoptosis and inhibited their stimulatory activity on allogeneic lymphocytes. These changes correlated with down-regulation of co-stimulatory and adhesion molecules such as CD40, CD54, CD80 and CD86. No differences were observed in mannose receptor (MR)-mediated endocytosis, measured by the uptake of fluorescein isothiocyanate (FITC)-dextran. MDDC differentiated in the presence of MMF showed significantly reduced maturation upon stimulation with lipopolysaccharide, as judged by lower expresson of CD83 and co-stimulatory molecules, lower production of tumour necrosis factor (TNF)-alpha, IL-10, IL-12 and IL-18 as well as lower stimulation of alloreactive T cells including naive CD4+ CD45RA+ T cells. In contrast, MDDC matured in the presence of MMF showed a more marked decrease in the FITC-dextran uptake than mature MDDC cultivated without MMF and the phenomenon correlated with down-regulation of the MR expression. These results suggest that MMF impairs differentiation, maturation and function of human MDDC in vitro, which is an additional mechanism of its immunosuppressive effect.

Interactions between leukocytes and epithelial cells may play a key role in Helicobacter pylori-associated gastric mucosal inflammation. This process is mediated by various cell adhesion molecules. The present study examined the molecular mechanisms leading to H. pylori-induced epithelial cell intercellular adhesion molecule-1 (ICAM-1; also called CD54) expression. Coculture of epithelial cells with cytotoxin-associated gene pathogenicity island-positive (cag PAI(+)) H. pylori strains, but not with a cag PAI(-) strain or H. pylori culture supernatants, resulted in upregulation of steady-state mRNA levels and cell surface expression of ICAM-1. Coculture with H. pylori induced an increase in luciferase activity in cells which were transfected with a luciferase reporter gene linked to the 5'-flanking region of the ICAM-1 gene. H. pylori activated the ICAM-1 promoter via the NF-kappaB binding site. An inducible nuclear protein complex bound to the ICAM-1 NF-kappaB site and was identified as the NF-kappaB p50-p65 heterodimer. H. pylori induced the degradation of IkappaB-alpha, a major cytoplasmic inhibitor of NF-kappaB, and stimulated the expression of IkappaB-alpha mRNA. Pretreatment of epithelial cells with pyrrolidine dithiocarbamate, which blocks NF-kappaB activation, inhibited H. pylori-induced ICAM-1 expression. THP-1 macrophagic cells, peripheral blood mononuclear cells, and purified neutrophils adhered to H. pylori-infected epithelial cells to a greater extent than to uninfected cells. These results show that H. pylori directly induces expression of ICAM-1 on gastric epithelial cells in an NF-kappaB-dependent manner that may support leukocyte attachment during inflammation.

OBJECTIVE

To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC.

METHODS

DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo.

RESULTS

DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P<0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P < 0.01).

CONCLUSIONS

H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.

Langerhans cells (LCs) represent an immature population of myeloid dendritic cells (DCs). As a result of their unique Birbeck granules (BGs), langerin expression, and heterogeneous maturation process, they differ from other immature DCs. Monocyte-derived LCs (MoLCs) mimic epidermal LCs. MoLCs with characteristic BGs are generated by culturing blood-derived monocytes with granulocyte macrophage-colony stimulating factor, interleukin (IL)-4, and transforming growth factor-beta1. Here, we compare maturation-induced antigen expression and cytokine release of LCs with MoLCs. To achieve comparable cell populations, LCs and MoLCs were isolated by CD1c cell sorting, resulting in high purity. In unstimulated cells, CD40 was expressed at equal levels. After stimulation with CD40 ligand (CD40L), LCs and MoLCs acquired CD83 and increased CD86. High CD80 expression was exclusively detected in CD1c-sorted MoLCs. Human leukocyte antigen-DR and CD54 expression was found in all cell populations, however, at different intensities. CD40 triggering increased the potency of LCs and MoLCs to stimulate CD4+ T cell proliferation. Activated MoLCs released IL-12p70 and simultaneously, anti-inflammatory IL-10. The application of the Toll-like receptor ligands peptidoglycan, flagellin, and in particular, lipopolysaccharide (LPS) increased the corelease of these cytokines. LCs secreted IL-10 at a comparable level with MoLCs but failed to produce high amounts of IL-12p70 after application of danger signals. These data indicate that MoLCs as well as LCs display no maturation arrest concerning CD83 and CD86 expression. In difference to MoLCs, LCs resisted activation by CD40L and LPS in terms of IL-12 production. This shows that natural and generated LCs share similar features but differ in relevant functions.