The liver has a role in T cell tolerance induction, which is mainly achieved through the functions of tolerogenic hepatic antigen-presenting cells (APCs) and regulatory T cells. Hepatic stellate cells (HSCs) are known to have various immune functions, which range from immunogenic antigen presentation to the induction of T cell apoptosis. Here we report a novel role for stellate cells in vetoing the priming of naive CD8 T cells. Murine and human HSCs and stromal cells (but not hepatocytes) prevented the activation of naive T cells by dendritic cells, artificial APCs, and phorbol 12-myristate 13-acetate/ionomycin by a cell contact-dependent mechanism. The veto function for inhibiting T cell activation was directly correlated with the activation state of HSCs and was most pronounced in HSCs from fibrotic livers. Mechanistically, high expression levels of CD54 simultaneously restricted the expression of interleukin-2 (IL-2) receptor and IL-2 in T cells, and this was responsible for the inhibitory effect because exogenous IL-2 overcame the HSC veto function.

CONCLUSIONS

Our results demonstrate a novel function of HSCs in the local skewing of immune responses in the liver through the prevention of local stimulation of naive T cells. These results not only indicate a beneficial role in hepatic fibrosis, for which increased CD54 expression on HSCs could attenuate further T cell activation, but also identify IL-2 as a key cytokine in mediating local T cell immunity to overcome hepatic tolerance.

CD40 is a member of the TNF family of receptors that has been shown to play a crucial role in enhancing dendritic cell activity and fostering anti-tumor immune responses. In this study, we demonstrate the in vitro properties and in vivo efficacious activity of the CD40 agonist antibody, CP-870,893. CP-870,893 is a fully human, IgG2 antibody that selectively interacts with CD40 at a site distinct from its ligand-binding region with a KD of 0.4 nM. It enhances the expression of MHC class II, CD54, CD86, and CD23 on human B cells in vitro. CP-870,893 also enhances dendritic cell activity as evidenced by cytokine secretion (IL-12, IL-23, IL-8), the upregulation of CD86 and CD83, and the ability to prime T cells to secrete IFNγ. In SCID-beige mice, a single parenteral injection of CP-870,893 was therapeutically effective against several CD40(pos) human tumors (B-cell lymphoma, breast, colon, and prostate) indicating direct effects on tumor cell survival and/or growth. When mice were co-implanted with human T cells and dendritic cells, the activity of CP-870,893 against CD40(pos) tumors increased, and efficacy was also observed against CD40(neg) and CD40(low) tumors demonstrating the ability of CP-870,893 to enhance anti-tumor immune function in vivo. These studies suggest that CP-870,893 has the potential to be efficacious against a wide range of tumor types through both direct and immune-mediated effects.

Based on a previous report on the effect of a matrix metalloproteinase (MMP) inhibitory compound, MMI270, in regulating tumor-induced angiogenesis, as well as recent findings concerning functional correlations among tumor metastasis, angiogenesis and lymphangiogenesis, we investigated the anti-metastatic efficacy of MMI270 in a murine model of lymph node metastasis of lung cancer, and analyzed whether this inhibitor could also regulate lymphangiogenesis-related properties of murine lymphatic endothelial cells (LECs) and invasive properties of Lewis lung cancer (LLC) cells. The observation that MMI270 led to a significant decrease in the weight of tumor-metastasized lymph nodes of mice led us to test its anti-lymphangiogenic and anti-invasive effects in vitro. Murine LECs were characterized by an in vitro tube formation assay, by semi-quantitative RT-PCR assay to examine the expression of mRNAs for flt-4, Flk-1, Tie-1, Tie-2, CD54/ICAM1, vWF, MMPs and uPA, and by western blotting to confirm the protein expression of flt-4 and CD31/PECAM. This is the first report on the expression of MMP-2, MMP-9 and MT1-MMP in murine LECs, as well as on the inhibition of their enzymatic activity, and of the invasive ability and tube-forming property of LECs by an MMP inhibitor. Furthermore, MMI270 was shown to strongly inhibit the activity of MMP-2 and -9 produced by LLC cells and the invasion of these cells through Matrigel. In summary, the present results indicate that MMI270, apart from its anti-tumor angiogenic application, might be useful as an anti-metastatic drug, on the basis of its downregulatation of both the lymphangiogenesis-related properties of LECs and the invasive properties of LLC cells in vitro.

The aim of this study is to optimize the experimental conditions for an in vitro skin sensitization test using the human cell lines THP-1 and U-937. As regards pre-culturing time, the expression of CD86 on DNCB-treated THP-1 cells tended to be higher after 48h and 72h pre-culture compared with other time points evaluated. Next, we investigated the effect of chemical treatment time, and found that induction of CD86 expression on THP-1 cells by DNCB reached a plateau after 24h. Augmentation of CD86 expression is often observed when cells are treated with a subtoxic dose of allergens. To determine the appropriate dose of test samples, the cytotoxicity of test samples to THP-1 and U-937 cells was assessed with MTT assay, and the 50% inhibitory concentration (IC50) of each test sample was calculated. Based on the cytotoxicity assay data, four concentrations in the range between toxic and non-toxic were selected (0.1x, 0.5x, 1x and 2x IC50). Several kinds of antibodies were tested for staining THP-1 and U-937 cells treated with allergens/non-allergens (e.g., DNCB, Ni/SLS), and suitable antibodies for staining CD86 and CD54 were selected. We confirmed that the working dilutions of the selected CD86 and CD54 antibodies were appropriate for use in our method. The effect of an FcR blocking procedure was also evaluated. The mean fluorescence intensity (MFI value) was decreased by the FcR blocking procedure, which indicated that non-specific staining was blocked. Therefore, this procedure should be included in the method. Based on our findings, the protocol for this assay was optimized and the experimental conditions to be used in a future validation study were identified. We propose to call this kind of in vitro skin sensitization test h-CLAT, which is short for human Cell Line Activation Test.

After application of haptens to the skin, Langerhans cells (LC), i.e. immature dendritic cells (DC) in the skin, move to secondary lymphoid organs to sensitize naive T cells. During this process, LC become mature DC with augmented expression of various co-stimulatory molecules and MHC class II antigens. In this scenario, however, critical questions remain as to what kind of chemicals can induce this maturation process through what kind of mechanisms. To clarify these questions, we used monocyte-derived CD1a+ DC instead of LC since LC maturated spontaneously in vitro culture. After we confirmed that monocyte-derived DC showed at least phenotypic characteristics and a response to TNF-alpha similar to LC, we added various chemicals, i.e., dinitrochlorobenzene (DNCB), trinitrochlorobenzene (TNCB), NiCl2, ZnCl2, sodium dodecyl sulfate (SDS), or benzalkonium chloride (BC), to a culture of purified monocyte-derived CD1a+ DC. Of these chemicals, only NiCl2 and DNCB significantly increased the surface expression of CD54, CD86, HLA-DR antigen, and interleukin (IL)-1 beta production, while SDS, BC, or ZnCl2 could not augment them, except for weak augmentation of CD86 expression by SDS. The increase in the expression of CD86 induced by NiCl2 or DNCB was most remarkable, being observed in DC from almost all the subjects we examined. TNCB could also induce responses similar to those induced with DNCB, but the number of subjects whose DC responded to it was far less than that of subjects whose DC responded to NiCl2 or DNCB. In spite of the augmented CD86 expression on DC treated with DNCB or NiCl2, these chemicals induced different responses of DC in their expression of CD54 and HLA-DR and the production of IL-6 and tumor necrosis factor (TNF)-alpha. In addition, the up-regulation of CD86 expression on DC treated with DNCB was significantly suppressed by either anti-IL-1 beta or anti-TNF-alpha antibody, while that by NiCl2 was relatively insensitive to these antibody treatments. Finally, the protein kinase C inhibitor, H7, but not staurosporine, could suppress the augmentation of CD86 expression on DC induced either by NiCl2 or by DNCB. These data suggest that DC respond to some haptens by changing their expression of several co-stimulatory molecules and their production of cytokines with a resultant change in antigen-presenting function. They also suggest that these chemicals stimulate DC by different mechanisms. By these responses, DC may modulate the final immune response to chemicals.

Transgenic mice overexpressing in B lymphocytes either Bcl-2 or a TNF receptor-associated factor (TRAF)2 mutant lacking the N-terminal RING and zinc finger domains located at the N terminus of the molecule (TRAF2DN), which mimics TRAF1, developed lymphadenopathy and splenomegaly due to polyclonal B cell expansion. Remarkably, TRAF2DN/Bcl-2 double-transgenic mice contained B cell populations similar to those observed in TRAF2DN mice. However, over time, they developed severe splenomegaly and lymphadenopathy, and most animals also developed leukemia, pleural effusion, and, in some cases, ascites associated with monoclonal and oligoclonal B cell neoplasms. The life span of TRAF2DN/Bcl-2 mice was markedly reduced compared with Bcl-2 and TRAF2DN single-transgenics or wild-type littermates. The expanded B cell population of TRAF2DN/Bcl-2 double-transgenic mice was primarily comprised of small/medium-size noncycling B220(M)/IgM(H)/IgD(L)/CD21(L)/CD23(NULL)/CD11b(+)/CD5+ cells that were Bcl-6-negative, consistent with a B-1 phenotype. The cells also expressed high levels of CD54 and other adhesion molecules. In vitro, these B cells showed comparable proliferation rates to those of wild-type counterparts but exhibited markedly increased survival and were resistant to apoptosis induced by chemotherapeutic agents and glucocorticoids. Histopathologic features were consistent with mouse small B cell lymphoma progressing to leukemia with many similarities to human chronic lymphocytic leukemia. Given that many human chronic lymphocytic leukemias overexpress TRAF1 and Bcl-2, our findings suggest that cooperation between Bcl-2 and TRAF pathways contributes to the development of this type of leukemia.

We investigated the phenotype and functional capacities of tumour-infiltrating lymphocytes (TIL), freshly isolated from primary renal cell carcinoma (RCC) specimens (n = 20). Three-colour flow cytometry immunophenotyping revealed that RCC TIL consist mainly of CD3+ T cells, with a clear predominance of CD4- CD8+ over CD4+ CD8- T cells, and a marked population of CD4+ CD8+ T cells. Natural killer (NK) cells were also strongly represented >> 25% in 15 of 20 tumour samples), while B cells constituted a minor TIL subset (< 5% in 18 of 20 tumour samples). More importantly, the T and NK cells within the tumour displayed a significantly higher expression of the early activation marker CD69 than their counterparts in adjacent normal renal tissue and in peripheral blood. Expression of CD54 and of HLA-DR was also elevated on CD3+ TIL, and HLA-DR expression was further vigorously up-regulated following ex vivo stimulation with anti-CD3, all suggesting enhanced immune activity within the tumour microenvironment. CD3+ CD4+ TIL displayed a normal capacity to up-regulate CD25 expression and to secrete both Th1-type (IL-2, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)) and Th2-type (IL-4, IL-5 and IL-10) cytokines upon triggering with anti-CD3. Furthermore, cytokine production was susceptible to modulation by CD28 costimulation. CD3+ CD8+ TIL, on the other hand, consistently demonstrated a poor up-regulation of CD25 upon triggering with anti-CD3, and displayed poor ex vivo cytolytic activity in an anti-CD3-redirected 4-h cytotoxicity assay against murine P815 cells. Collectively, our findings indicate that the CD3+ CD4+ TIL in RCC have normal functional capacities, whereas the proportionally major CD3+ CD8+ TIL are functionally impaired. The relevance of these findings to the in vivo local immune response in RCC is discussed.

Most bacteria that enter the bloodstream are taken up by the liver. Previously, we reported that such organisms are initially bound extracellularly and subsequently killed by immigrating neutrophils, not Kupffer cells as widely presumed in the literature. Rather, the principal functions of Kupffer cells demonstrated herein are to clear bacteria from the peripheral blood and to promote accumulation of bactericidal neutrophils at the principal site of microbial deposition in the liver, i.e., the Kupffer cell surface. In a mouse model of listeriosis, uptake of bacteria by the liver at 10 min postinfection i.v. was reduced from approximately 60% of the inoculum in normal mice to approximately 15% in mice rendered Kupffer cell deficient. Immunocytochemical analysis of liver sections derived from normal animals at 2 h postinfection revealed the massive immigration of neutrophils and their colocalization with Kupffer cells. Photomicrographs of the purified nonparenchymal liver cell population derived from these infected mice demonstrated listeriae inside neutrophils and neutrophils within Kupffer cells. Complementary adhesion molecules promoted the interaction between these two cell populations. Pretreatment of mice with mAbs specific for CD11b/CD18 (type 3 complement receptor) or its counter-receptor, CD54, inhibited the accumulation of neutrophils in the liver and the elimination of listeriae. Complement was not a factor; complement depletion affected neither the clearance of listeriae by Kupffer cells nor the antimicrobial activity expressed by infiltrating neutrophils.

Tumor-infiltrating CD8+ T-lymphocytes (T-TILs) are thought to be relevant to immunosurveillance of several tumor types including B-cell non-Hodgkin's lymphoma. B- and T-lymphocyte interactions via cellular adhesion molecules (CAMs), recognition molecules (HLAs), and costimulatory molecules (CSMs) are necessary for optimal antigen-specific T-cell activation to occur and may be important in generating effective host T-TIL responses. We previously found that low T-TIL response (CD8+ T cells < 6%) correlates with statistically shorter relapse-free survival in patients with diffuse large-cell lymphoma (DLCL). We now extend our observations in 71 DLCL patients by analyzing malignant B-cell expression of the following molecules important in T-cell activation: (a) recognition molecules [MHC I (MAS and MCA) and MHC II (HLA-DR, -DP, -DQ)]; (b) CAMs [leukocyte function antigen 1 (CD11a and CD18) and intracellular adhesion molecule 1 (CD54)]; and (c) CSMs [B7.1 (CD80) and B7.2 (CD86)]. Eighteen patients (25%) had low a T-TIL response, and 53 patients (75%) had a high T-TIL response. Overall, expression of the MHC class H molecules HLA-DR and HLA-DQ was most conserved. The loss of B7.2 (P = 0.04), intracellular adhesion molecule 1 (P = 0.0004), MAS (P = 0.02), and HLA-DR (P = 0.0004) expression was significantly associated with decreased T-TIL response. In 100% of patients with low T-TIL responses, at least one HLA, CAM, or CSM was undetectable on the malignant B cells by immunohistochemical staining (mean number of molecules lost = 2.67). In contrast, 49% of patients with high T-TIL responses had no losses in HLA, CAM, or CSM expression (mean number of molecules lost = 0.89). The mean number of absent molecules (HLA, CAM, or CSM) was significantly associated with T-TIL response (P = 0.0001). We conclude that loss of HLA, CAM, or CSM expression on malignant B cells is associated with a poor host T-cell immune response. In addition, because patients with low T-TIL response had lost expression of multiple cellular adhesion, recognition, and costimulatory molecules, our results suggest that a combination of immunorestorative therapies may be required to generate effective antitumor T-cell responses in B-cell DLCL.

Arsenic trioxide (As2O3) is used clinically to treat acute promyelocytic leukemia and has activity in vitro against several solid tumour cell lines, where induction of differentiation and apoptosis are the prime effects. To investigate the potential therapeutic application of As2O3 to breast cancer, we analysed the effects of As2O3 on the growth of four human breast cancer cell lines: MCF7, MDA-MB-231, T-47D and BT-20. Cells were cultured in 0.5, 2 and 5 microM AS2O3, a range of pharmacologically achievable concentrations of AS2O3. At>> or = 2 microM, AS2O3 rapidly induced cell death by apoptosis in MCF7 and MDA-MB-231 while T-47D and BT-20 were partially resistant. At 0.5 microM, As2O3 was subapoptotic but induced features of differentiation consisting in upregulation of ICAM-1 (CD54), a marker of mammary epithelial differentiation, and cell cultures appeared morphologically more organized. Furthermore, we demonstrate by standard cytotoxicity assays that As2O3 treatment can augment breast cancer cell lysis by lymphokine-activated killer cells and demonstrate an important role of the ICAM-1/LFA-1 interaction in this process. This additional activity of As2O3 could translate into improved antitumour immunosurveillance in vivo. In conclusion, As2O3 induced varying degrees of differentiation, apoptosis and lysis in these model cell lines, and may be a promising adjuvant to current treatments of breast cancer by virtue of its triple apoptotic, differentiative and immunomodulatory effects.

The blood-retinal barrier (BRB), which is composed of the retinal pigment epithelium (RPE) and retinal vascular endothelium, normally restricts the traffic of lymphocytes into the retina. During ocular inflammatory conditions such as posterior uveitis there is a large increase in lymphocyte migration across the BRB. The differential role played by the two barrier sites, however, remains unclear. To evaluate the role of the posterior BRB, the migration of CD4+ antigen-specific T-cell line through rat RPE cell monolayers was investigated in vitro using time-lapse videomicroscopy. The adhesion molecules involved in controlling transepithelial migration across normal and interferon-gamma (IFN-gamma)-activated RPE was assessed with monoclonal antibodies directed against cell adhesion molecules. Lymphocytes were treated with antibodies specific for CD11a (alpha L subunit of LFA-1), CD18 (beta 2 subnit of the leucam family) and CD49 d (alpha 4 subnit of very late activation antigen-4, VLA-4), and the RPE with antibodies specific for CD54 (intracellular adhesion molecule-1, ICAM-1) and CD 106 (vascular cell adhesion molecule-1, VCAM-1). Migration across unstimulated RPE was inhibited by antibodies to ICAM-1 (48.6 +/- 3.5% reduction), leucocyte functional antigen-1 (LFA-1) alpha (61 +/- 5.2%) and LFA-1 beta (63.2 +/- 4.7%), but not by antibodies to VLA-4. VCAM-1 was not expressed on untreated RPE. Following activation of the RPE monolayers for 72 hr with IFN-gamma, antibodies to LFA-1 alpha, LFA-1 beta and ICAM-1 inhibited migration by 49.9 +/- 9.4%, 63.6 +/- 5.5% and 47.7 +/- 4.2% respectively. Antibodies to VLA-4 and VCAM-1 blocked migration by 21.5 +/- 8.4% and 32.3 +/- 6.2%, respectively, which correlated with the induction of VCAM-1 expression on RPE and increased migration. Under these conditions blocking both VCAM-1 and ICAM-1 reduced migration by 70.9 +/- 2.3%, which was greater than the effect of blocking either of these molecules alone. These results demonstrate that the posterior barrier of the BRB utilizes the same principle receptor-ligand pairings in controlling lymphocyte traffic into the retina as the vascular endothelium of the anterior BRB.

BACKGROUND

Adhesion of haematopoietic progenitor cells (HPC) to human bone marrow endothelial cells (HBMEC) plays a key role in homing of HPC to bone marrow. Here we describe four new HBMEC cell lines that can be used to study the (specific) adhesion of HPC to HBMEC.

METHODS

HBMEC were immortalised with a retroviral construct containing the human papilloma virus 16 E6/E7 genes. Four cell lines were characterised.

RESULTS

The cell lines showed their endothelial nature by the expression of von Willebrand Factor and VE-cadherin (CD144). Electron microscopic analysis revealed normal endothelial-cell characteristics, including the presence of Weibel-Palade bodies and intercellular junction structures. An extensive phenotypic analysis of the cell-lines was performed, they were found to resemble primary HBMEC. The only difference found was the absence of expression of E-selectin (CD62e) and VCAM-1 (CD106) on resting HBMEC cell lines. Upon stimulation with IL-1beta the expression of E-selectin, VCAM-1 and ICAM-1 (CD54) was upregulated. All resting cell lines bound CD34+ HPC. Adhesion was increased by addition of the phorbol ester PMA. Two cell lines showed increased binding upon IL-1beta prestimulation. Highest adhesion was observed after the combination of IL-1beta prestimulation of the endothelial cells and addition of PMA. Binding of CD34+ HPC to HBMEC was compared with the binding to human umbilical vein endothelial cell lines and to a human dermal microvascular endothelial cell line (HMEC-1). So far, we have only found relatively less binding of HPC to IL-1beta prestimulated HMEC-1 cells, which could be explained by a reduced induction of E-selectin and VCAM-1 upon IL-1beta stimulation of these cells.

CONCLUSIONS

The immortalised HBMEC cell lines have maintained their normal phenotype for the majority of characteristics examined. The expression of E-selectin and VCAM-1, which are not constitutively expressed on the cell lines, can be induced by stimulation of the endothelial cells with IL-1beta. The cell lines have furthermore maintained their capability to bind HPC. They will therefore be useful to investigate the interactions between HPC and HBMEC involved in homing of HPC.

Patients with low-grade myelodysplastic syndromes (MDS) show high levels of tumor necrosis factor α (TNFα) and up-regulation of apoptosis in the marrow. In contrast, marrow cells in advanced MDS are typically resistant to TNFα-induced apoptosis but are rendered apoptosis-sensitive on coculture with stroma. The present studies show that CD34(+) marrow cells in advanced MDS express high levels of TWIST, a basic helix-loop-helix transcription factor that opposes p53 function. TWIST levels correlated with disease stage (advanced>> low grade; P = .01). Coculture with HS5 stroma resulted in down-regulation of TWIST and increased apoptosis in response to TNFα in CD34(+) cells from advanced MDS; the same effect was achieved by TWIST-specific RNA interference in CD34(+) cells. In primary MDS marrow stroma TWIST expression was lower than in healthy controls; suppression of TWIST in stroma interfered with induction of apoptosis sensitivity in cocultured CD34(+) cells. Stroma cells so modified expressed reduced levels of intercellular adhesion molecule-1 (ICAM1; CD54); blockade of ICAM1 in unmodified stroma was associated with reduced apoptosis in cocultured CD34(+) MDS marrow cells. These data suggest role for dysregulation of TWIST in the pathophysiology of MDS. Conceivably, TWIST or components in the signaling pathway could serve as therapeutic targets for patients with MDS.

BACKGROUND

The fact that Pityrosporum ovale plays a part in seborrhoeic dermatitis is well established but the mechanism of this relationship has not been established.

OBJECTIVE

To compare the number and type of inflammatory cells and mediators in skin biopsies from normal and lesional skin from the trunk and scalp in patients with seborrhoeic dermatitis, Pityrosporum (Malassezia) folliculitis and in normal skin from healthy controls.

METHODS

The skin biopsies were stained using the labelled Streptavidin-biotin

METHODS

The following markers were studied: CD4, CD8, CD68, HLA-DR, NK1, CD16, C1q, C3c, IgG, CD54 (ICAM-1), interleukin (IL) -1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, tumour necrosis factor-alpha and interferon-gamma.

RESULTS

HLA-DR+ cells were seen in the highest number, and were higher in lesional skin compared with normal skin from both patients and healthy volunteers. ICAM-1 expression was also increased in lesional skin. C1q and the interleukins showed an increased cellular and intercellular staining in patients compared with healthy controls and the intercellular staining was often more intense in lesions compared with non-lesional skin. Staining was often more intense when Malassezia (Pityrosporum ovale) yeast cells were present.

CONCLUSIONS

An increase in NK1+ and CD16+ cells in combination with complement activation indicates that an irritant non-immunogenic stimulation of the immune system is important. The result with the interleukins showed both an increase in the production of inflammatory interleukins as well as in the regulatory interleukins for both TH1 and TH2 cells. Similarities to the immune response described for Candida albicans infections indicate the role of Malassezia in the skin response in seborrhoeic dermatitis and Pityrosporum folliculitis.

Increasing evidence suggests the existence of polarized human T cell responses described as Th1-type (promoting cell-mediated immunity) and Th2-type (promoting humoral immunity), characterized by a dominant production of either interferon-gamma (IFN-gamma) or IL-4, respectively. Little is known about the intratumoural activation of infiltrating lymphocytes (TIL) in human gliomas. Therefore, we assessed fresh TIL at cellular and molecular levels to find out if they were activated and polarized into a type 1 or 2 immune response. Flow cytometry analysis of TIL revealed that the major subset was made of T lymphocytes. Double labelling with alpha-CD3 and adhesion/ activation markers revealed T cell subsets expressing CD49a, CD49b, CD54, and CD15, some of which were almost absent in autologous T peripheral blood lymphocytes (T-PBL). Furthermore, the proportions of T-TIL expressing CD56, CD65, or CD25 were several-fold higher than in T-PBL. Intratumoural functional activation of TIL was tested by semiquantitative assessment in relative units (RU) of lymphokine gene activation with mRNA reverse transcriptase-polymerase chain reaction (RT-PCR). All TIL populations except one significantly expressed IL-4 1 to 2 logs of RU above healthy PBL baseline. Similarly, all patients expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) in a range comparable to IL-4. However, most TIL populations did not express IFN-gamma, IL-2, and tumour necrosis factor-beta (TNF-beta) at higher levels than healthy normal PBL. The increase proportion of T cells expressing activation markers and the consistent detection of significant IL-4 and GM-CSF lymphokine gene activation in TIL populations suggested a predominant type 2 intratumoural immune response that does not promote cell-mediated tumouricidal activity and may contribute to the inefficiency of the antiglioma immune response.

BACKGROUND

Endothelial cells are important in initiating adhesive processes between circulating cells and extracellular structures and changes in their distribution are believed to be important in many pathologic conditions. Since little is known about the detailed distribution of adhesion molecules in human endothelium in different sites and circumstances, the present study has undertaken a detailed analysis of 5 of the putative most important adhesion molecules on a wide range of normal tissue endothelium. We have compared this reactivity with that seen in a comprehensive range of vascular tumors both benign and malignant.

METHODS

Fresh samples of a wide range of normal tissues and vascular tumors were stained by antibodies against the following adhesion molecules; intercellular adhesion molecule-1 (CD54), E-selectin (endothelial cell adhesion molecule-1), vascular cell adhesion molecule-MUC-1, P-selectin (platelet activation-dependent granule to external membrane protein, CD62) and MUC-18 using either the APAAP immuno-alkaline phosphatase or an immunoperoxidase method.

RESULTS

Labeling of the endothelial cells in different normal tissues with intercellular adhesion molecule-1, P-selectin, and MUC-18 was heterogeneous both in terms of vessel size and strength of staining. Vascular cell adhesion molecule-1 and E-selectin were largely absent. The vascular tumors were likewise variable in their staining patterns which frequently differed from the immunophenotype of the reactive vessels surrounding the tumor.

CONCLUSIONS

This study demonstrates that the expression of adhesion molecules of the immunoglobulin and selectin family on normal tissue endothelium, and vascular tumors is much less predictable than that obtained with other vascular markers such as F8 RA, CD31, CD34, and CD36. Adhesion molecules show considerable heterogeneity of expression on vascular endothelium which presumably reflects their varied functions on different types of vessel. In general their expression is markedly reduced on vascular tumors.

Activation of T cells often requires both activation signals delivered by ligation of the TCR and those resulting from costimulatory interactions between certain T cell surface accessory molecules and their respective counter-receptors on APC. CD11a/CD18 complex on T cells modulate the activation of T cells by interacting with its counter-receptors intracellular adhesion molecule (ICAM-1) (CD54) and/or ICAM-2 on the surface of APC. The costimulatory ability of ICAM-1 has been demonstrated. Using a soluble ICAM-2 Ig fusion protein (receptor globulin, Rg) we demonstrate the costimulatory effect of ICAM-2 during the activation of CD4+ T cells. When coimmobilized with anti-TCR-1 mAb ICAM-2 Rg induced vigorous proliferative response of CD4+ T cells. This costimulatory effect of ICAM-2 was dependent on its coimmobilization with mAb directed at the CD3/TCR complex but not those directed at CD2 or CD28. Both resting as well as Ag-primed CD4+ T cells responded to the costimulatory effects of ICAM-2. The addition of mAb directed at the CD11a or CD18 molecules almost completely inhibited the responses to ICAM-2 Rg. These results are consistent with the role of CD11a/CD18 complex as a receptor for ICAM-2 mediating its costimulatory effects. Stimulation of T cells with coimmobilized anti-TCR-1 and ICAM-2 resulted in the induction of IL-2R (CD25), and anti-Tac (CD25) mAb inhibited this response suggesting the contribution of endogenously synthesized IL-2 during this stimulation. These results demonstrate that like its homologue ICAM-1, ICAM-2 also exerts a strong costimulatory effect during the TCR-initiated activation of T cells. The costimulatory effects generated by the CD11a/CD18:ICAM-2 interaction may be critical during the initiation of T cell activation by ICAM-1low APC.

In order to analyse immune-stimulatory effects of infection of human tumor cells with Newcastle Disease Virus (NDV), gamma-irradiated human breast carcinoma, colon-carcinoma or glioblastoma cells from defined cell lines were modified either by true infection with live virus or by cell surface adsorption of UV-inactivated replication deficient virus. Modification with live but not inactive NDV induced in all human tumor cells IFN-beta and the chemokines RANTES and IFN-gamma-inducible protein-10 (IP-10). In addition, infection by live NDV induced upregulation of HLA-ABC-molecules in all tumor lines tested and HLA-DR molecules in breast carcinoma lines. Two cell adhesion molecules, ICAM-I (CD54) and LFA-3 (CD58), were also upregulated on human tumor cells after infection with live NDV. When infection of MCF-7 breast carcinoma cells by NDV was performed in the presence of neutralizing anti-IFN-beta antibodies no upregulation of HLA molecules was observed suggesting an important role of IFN-beta in this process. Forty-eight to 72 hours after infection of the irradiated tumor cells with live NDV, many tumor cells were dead or in early or late stages of apoptosis. These results provide explanations for the function of the virus-modified autologous tumor vaccine ATV-NDV with which promising clinical results have already been obtained.

Abnormal immunity and its related complications are the major causes of mortality and morbidity in diabetes patients. Macrophages, as one of the important innate cells, play pivotal roles in controlling immune homeostasis, immunity, and tolerance. The effects of hyperglycemia on the function of macrophages in hosts remain to be determined. Here we used mice with streptozotocin (STZ)-induced diabetes for long term to study the changes of macrophages. We found that F4/80(+) peritoneal exudate macrophages (PEMs) from mice with diabetes for 4 months displayed significantly reduced CD86 and CD54 expression and tumor necrosis factor (TNF)-α and IL-6 production but enhanced nitric oxide (NO) secretion compared with control mice when treated with interferon (IFN)-γ and lipopolysaccharide (LPS), while the activity of arginase in PEMs from diabetic mice was significantly higher than control mice when stimulating with IL-4. These dysfunctions of macrophages could be efficiently reversed by insulin treatment. Importantly, in vitro bone marrow-derived macrophages showed similar functional changes, indicating the epigenetic alteration of macrophage precursors in these mice. In an in vitro culture system, high glucose and insulin significantly altered TNF-α, IL-6, and NO production and arginase activity of macrophages, which was reversed by the treatment with AKT and ERK inhibitors. Therefore, hyperglycemia and insulin deficiency can modify macrophage function through AKT-mTOR and ERK pathways and through epigenetic effects on macrophage precursors. To further identify different components of diabetes on the dysfunction of macrophages is important for efficient prevention of diabetic complications.

The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to interact with other cells. PMN activation with various stimuli mobilizes intracellular sialidase to the plasma membrane, where it cleaves sialic acid from cell surfaces. Because enhanced PMN adherence, spreading, deformability, and motility each are associated with surface desialylation and are critical to PMN diapedesis, we studied the role of sialic acid on PMN adhesion to and migration across pulmonary vascular endothelial cell (EC) monolayers in vitro. Neuraminidase treatment of either PMN or EC increased adhesion and migration in a dose-dependent manner. Neuraminidase treatment of both PMNs and ECs increased PMN adhesion to EC more than treatment of either PMNs or ECs alone. Moreover, neuraminidase treatment of ECs did not change surface expression of adhesion molecules or release of IL-8 and IL-6. Inhibition of endogenous sialidase by either cross-protective antineuraminidase antibodies (45.5% inhibition) or competitive inhibition with pseudo-substrate (41.2% inhibition) decreased PMN adhesion to ECs; the inhibitable sialidase activity appeared to be associated with activated PMNs. Finally, EC monolayers preincubated with activated PMNs became hyperadhesive for subsequently added resting PMNs, and this hyperadhesive state was mediated through endogenous PMN sialidase activity. Blocking anti-E-selectin, anti-CD54 and anti-CD18 antibodies decreased PMN adhesion to tumor necrosis factor-activated ECs but not to PMN-treated ECs. These data implicate desialylation as a novel mechanism through which PMN-EC adhesion can be regulated independent of de novo protein synthesis or altered adhesion molecule expression. The ability of activated PMNs, through endogenous sialidase activity, to render the EC surface hyperadherent for unstimulated PMNs may provide for rapid amplification of the PMN-mediated host response.

Several immunoregulatory mechanisms are proposed to be effective both in human and experimental Trypanosoma cruzi infection. However, the role of CD4+CD25high T cells in Chagas disease has not yet been elucidated. These cells are critical for the regulation of immune response to infectious agents and in the control of autoimmune diseases. In this study, the presence of CD4+CD25high regulatory T cells in the whole blood of non-infected individuals (NI), and patients with the indeterminate (IND) and cardiac form (CARD) of Chagas disease was evaluated. To further characterize this population of regulatory cells, the co-expression of CTLA-4, CD62L, CD45RO, CD45RA, HLA-DR, CD40L, CD69, CD54, IL-10R and the intracellular molecules FOXP3 and IL-10 on the CD4+CD25high T lymphocytes was examined. FOXP3 was expressed by the majority of CD4+CD25high when compared with the other CD4+ T cells subsets in patients with Chagas disease. Patients with the IND form of the disease had a higher frequency of circulating regulatory CD4+CD25high T cells than patients with the CARD form. Moreover, there was an increase in CD4+CD25highFOXP3+ cells that were also IL-10+ in the IND group whereas, in the CARD group, there was an increase in the percentage of CD4+CD25high FOXP3+ cells that expressed CTLA-4. These data suggest that IL-10 produced by regulatory T cells is effective in controlling disease development in patients with the IND form. However, in individuals with the CARD form of the disease, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is not sufficient to control the progression of the disease. The data suggest that CD4+CD25highFOXP3+ regulatory T cells in patients with Chagas disease might play a role in the immune response against T. cruzi infection although with distinct effects in patients with the IND and CARD forms of disease.

OBJECTIVE

Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines.

METHODS

Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence.

RESULTS

All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2.

CONCLUSIONS

These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

Hyaluronan (HA), in the bone marrow stroma, is the major non-protein glycosaminoglycan component of extracellular matrix (ECM) involved in cell positioning, proliferation, differentiation as well as in receptor-mediated changes in gene expression. Repair of bone and regeneration of bone marrow is dependent on ECM, inflammatory factors, like chemokines and degradative factors, like metalloproteinases. We analyzed the interaction between human mesenchymal stem cells (h-MSCs) and a three-dimensional (3-D) HA-based scaffold in vitro. The expression of CXC chemokines/receptors, CXCL8 (IL-8)/CXCR1-2, CXCL10 (IP-10)/CXCR3, CXCL12 (SDF-1)/CXCR4, and CXCL13 (BCA-1)/CXCR5, and metalloproteinases/inhibitors MMP-1, MMP-3, MMP-13/TIMP-1 were evaluated in h-MSCs grown on plastic or on HA-based scaffold by Real-time PCR, ELISA, and immunocytochemical techniques. Moreover, the expression of two HA receptors, CD44 and CD54, was analyzed. We found both at mRNA and protein levels that HA-based scaffold induced the expression of CXCR4, CXCL13, and MMP-3 and downmodulated the expression of CXCL12, CXCR5, MMP-13, and TIMP-1 while HA-based scaffold induced CD54 expression but not CD44. We found that these two HA receptors were directly involved in the modulation of CXCL12, CXCL13, and CXCR5. This study demonstrates a direct action of a 3-D HA-based scaffold, widely used for cartilage and bone repair, in modulating both h-MSCs inflammatory and degradative factors directly involved in the engraftment of specific cell types in a damaged area. Our data clearly demonstrate that HA in this 3-D conformation acts as a signaling molecule for h-MSCs.

The CD7 molecule, one of the earliest T-lymphocyte Ag expressed during ontogeny, has recently been demonstrated to facilitate activation of T cells and to preferentially activate TCR-gamma/delta + subset of T cells. The CD7 Ag is also expressed on human NK cells, but its function has not been determined. In this study, expression and function of CD7 Ag on highly enriched NK cells (94 +/- 3% mean +/- SD, n = 12) obtained by negative selection from peripheral blood of normal donors were investigated. The CD7 Ag was found to be expressed at a significantly (p < 0.002) higher level on fresh NK cells than on IL-2-activated, NK cells. CD7 on human NK cells was found to be a signal-transducing molecule with a rapid increase in cytoplasmic free calcium observed on binding of anti-CD7 mAb to the surface of NK cells. Cross-linking of CD7 induced expression of surface activation molecules such as CD25, CD71, HLA-DR, CD69, and CD54. Activation by anti-CD7 mAb cross-linked to plastic or through goat anti-mouse Ig also induced a variety of NK cell functions: it stimulated secretion of IFN-gamma, led to proliferation of NK cells, as measured by [3H]thymidine incorporation, and significantly enhanced cytotoxicity of NK cells against K562 targets (p < 0.03). However, CD7 on NK cells did not seem to transduce a lytic signal, because it neither mediated redirected killing of Fc gamma R+ murine mastocytoma P815 cells nor triggered lysis of a hybridoma expressing the antibody in a membrane-bound form. CD7 molecules appeared to have a regulatory role in adhesion of NK cells to fibronectin, because cross-linking of CD7 on resting NK cells significantly augmented their adhesion to fibronectin-coated plastic surfaces. However, this induced adhesion was not associated with increased expression of beta 1-integrins on NK cells. Thus, CD7-mediated signals appear to augment function of adhesion molecules on NK cells, which may be involved in NK cell activation by providing both anchorage and costimulatory triggering.