In bronchial asthma, eosinophils found in the airways have an enhanced inflammatory capacity. We hypothesized that, at least in part, changes in functional phenotype are due to the effect of transendothelial migration. To model in vivo eosinophil trafficking to the lung, we cultured human pulmonary microvascular endothelial cell (HPMEC) monolayers on Transwell filters. The HPMECs were activated with interleukin (IL)-1beta to increase cell expression of intercellular adhesion molecule (ICAM)-1 and, hence, eosinophil transmigration. Peripheral blood eosinophils from allergic patients were added to HPMEC-covered Transwell filters and incubated for 3 h at 37 degrees C. The eosinophils were collected from below (migrated cells) and above (nonmigrated cells) the HPMEC monolayer to determine surface receptor expression, in vitro survival, and oxidative burst. Eosinophils never exposed to HPMECs were used as controls. Eosinophil cell surface expression of CD69, human leukocyte-associated antigen-DR (HLA-DR), and CD54 (ICAM-1) was significantly increased after transendothelial migration through IL-1beta-treated HPMECs compared with control cells (CD69: P<0.0005; HLA-DR and CD54: P<0.05) and nonmigrated eosinophils (CD69 and HLA-DR: P<0.05). Moreover, the percent in vitro survival (48 h) of migrated eosinophils was also significantly greater (P<0.0001 by trypan blue exclusion, P< 0.05 by flow cytometry) than that of control or nonmigrated eosinophils. Prolonged survival of migrated eosinophils was inhibited by addition of anti-granulocyte macrophage colony-stimulating factor (GM-CSF) antibodies (P<0.05) to the 48-h survival culture, suggesting that autocrine production of GM-CSF was, at least partially, responsible for increased eosinophil survival. Although GM-CSF protein was not measurable in survival culture supernates, GM-CSF messenger RNA (mRNA) was expressed in both nonmigrated and migrated eosinophils but not in control cells. Similarly, the eosinophils' oxidative burst induced by platelet-activating factor, formylmethionyl leucylphenylalanine, or phorbol myristate acetate was equally, and significantly, increased in both nonmigrated and migrated eosinophils (P<0.05 versus control). Therefore, whereas exposure of eosinophils to cytokine-activated HPMECs can increase surface receptor expression, in vitro survival, GM-CSF mRNA, and the respiratory burst, transendothelial migration can further potentiate receptor expression and survival in migrated cells. These results suggest that the process of transendothelial migration selectively participates in determining the eventual phenotype of airway eosinophils.

Recently we reported the expression of the human natural killer cell associated antigen CD56 (Leu 19/NKH1) in plasma cells of a majority of multiple myeloma (MM) patients. CD56 is known to be an isoform of the human neural adhesion molecule N-CAM which is involved in homotypic adhesive interactions. By immunophenotyping using four CD56 specific monoclonal antibodies and immunoprecipitation analysis we here confirm that the Leu 19 antigen expressed by myeloma plasma cells is identical to N-CAM and corresponds to the 145 kDa isoform. Because of the possible biological role of adhesion molecules on myeloma cells, we compared the expression of N-CAM with the intercellular adhesion molecule 1 (ICAM-1) and the beta 1 and beta 2 integrins. By immunogold-silver staining of cytospin preparations of mononuclear cell suspensions, bone marrow plasma cells of 17 MM patients were analysed. Plasma cells expressed N-CAM (CD56) in 14 patients. ICAM-1 (CD54) in 16 patients, and beta 2 integrins (CD18) in eight patients. beta 1 integrins (CD29) were expressed in all patients. The expression of beta 2 integrins was always very weak while N-CAM, ICAM-1 and the beta 1 integrins showed a moderate to strong positivity. The plasma cells of five haematological normal individuals lacked significant N-CAM expression but were positive for ICAM-1 and both integrin subgroups. One plasma cell leukaemia patient and two out of four end-stage MM patients showed no expression of N-CAM or beta 2 integrins on their circulating plasma cells. Among 11 previously established myeloma cell lines, surface expression of ICAM-1 and the integrins was detected in most cases, while N-CAM was present in only four lines. Most cell lines showed coexpression of the fibronectin receptors (VLA-4 and VLA-5) and the laminin receptor (VLA-6). The collagen receptor (VLA-2) was not expressed. The N-CAM negative cell lines included four cell lines that were derived from plasma cell leukaemia patients. These results indicate that the expression of adhesion molecules is an intrinsic part of the biology of multiple myeloma.

The outcome of immune responses can be predicted by the lymphokine production pattern of the participating cells. Cytokines of the T helper type 1 (Th1) cells mediate inflammatory responses and delayed-type hypersensitivity (DTH), whereas Th2-like T cells predominantly produce cytokines, which stimulate antibody production by B cells. Immunoregulatory therapy of autoimmune diseases with unknown antigens may be achieved by inhibiting the production of inflammatory cytokines and induction of protective cytokines of Th2-like T cells. To determine the immunoregulatory capacity of the phosphodiesterase inhibitor pentoxifylline (PTX), which is known to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), this drug was used in mitogen and antigen-stimulated lymphocyte cultures as well as in patients with multiple sclerosis. PTX significantly decreased TNF-alpha and interleukin-12 (IL-12), whereas it increased IL-4 and IL-10 production. In addition, PTX inhibited cell proliferation, which was associated with a marked reduction in CD25 (IL-2 receptor alpha-chain) and CD54 (intercellular adhesion molecule-1; ICAM-1) expression. Increasing doses of PTX significantly reduced TNF-alpha and IL-12 mRNA expression of blood mononuclear cells, but increased IL-4 and IL-10 expression in eight patients with relapsing-remitting multiple sclerosis. These results indicate that PTX modulates immune reactions favouring a Th2-like response and may therefore be useful for the treatment of autoimmune diseases with a dominant Th1-like T cell response.

The anaphylatoxin C3a is an important inflammatory mediator in the innate and adaptive immune systems. Recent reports in various animal models have fostered the role of C3a in mediating allergic reactions such as pulmonary allergies. However, data in humans are limited and the cellular targets for C3a are not fully understood. We sought to explore human dendritic cells as a new target for C3a, because C3a receptor (C3aR) expression has been described on myeloid cells, and dendritic cells are likely make contact with C3a at sites of inflammatory reactions. In this study, we demonstrated the expression of the C3aR on human monocyte-derived dendritic cells (MoDC) and its up-regulation by interferon (IFN)-alpha, IFN-gamma and prostaglandin E2 (PGE2). The strongest up-regulation was yielded by the combination of IFN-alpha+ IFN-gamma. Tumour necrosis factor-alpha (TNF-alpha) down-regulated the C3aR. After up-regulation of the C3aR by IFN-alpha+ IFN-gamma, C3a significantly up-regulated the surface expression of CD54, CD83 and CD86, but not of CD40, CD80 or human leucocyte antigen (HLA)-DR. C3a had no effect on the production of interleukin (IL)-10 or IL-12p70, or on the capacity of MoDC to stimulate autologous T-cell proliferation. However, C3a had a direct migratory effect on MoDC, as indicated by the induction of F-actin polymerization and migration in Boyden chamber experiments, which was pronounced after up-regulation of the C3aR with IFN-alpha+ IFN-gamma. Therefore, dendritic cells represent another group of target cells that might be recruited by C3a to areas of inflammation, in particular under conditions where IFNs are increased in the surrounding environment.

OBJECTIVE

The aim of the present study was to investigate the effect of a mixture of proteolytic enzymes (comprising trypsin, chymotrypsin and papain) on the metastatic model of syngeneic melanoma B16.

METHODS

140 C57B16 mice were divided into two control and two "treated" groups. Control groups received saline rectally, twice a day starting 24 h after intracutaneous transplantation (C1) or from the time point of the primary B16 melanoma extirpation (C2), respectively. "Treated" groups were rectally administered a mixture of 0.2 mg trypsin, 0.5 mg papain, and 0.2 mg chymotrypsin twice daily starting 24 h after transplantation (E1) or after extirpation of the tumor (E2), respectively. Survival of mice and B16 melanoma generalization were observed for a period of 100 days. Immunological evaluation of B16 melanoma cells in the ascites was accomplished. CD44, CD54 and CD106 cells were measured by flow cytometry.

RESULTS

Administration of proteolytic enzymes to mice inhibited the growth of primary tumors, and tumor recurrences were less numerous. Importantly, metastasis was considerably curtailed both in the vicinity of the primary tumor and at distant locales. These findings correlated with a decreased expression of CD44 and CD54 molecules in tumors exposed to proteolytic enzymes in vivo.

CONCLUSIONS

Our data suggest that serine and cysteine proteinases suppress B16 melanoma, and restrict its metastatic dissemination in C57B16 mice.

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. Extracorporeal photochemotherapy (ECP) has been introduced as an alternative treatment for GVHD refractory to conventional immunosuppressive treatment, although its mechanism of action is not yet clear. We investigated, in seven GVHD patients, the effects of ECP on dendritic cell maturation and cytokine production in an in vitro model that could mimic the potential in vivo effect of reinfusion of ECP-treated peripheral blood mononuclear cells. The model was based on co-culture of ECP-treated lymphocytes with monocyte-derived dendritic cells (DCs) of the same patient. We found that the co-culture of ECP-treated lymphocytes with immature DCs reduced CD54, CD40 and CD86 mean fluorescence intensity (MFI) significantly after lipopolysaccharide (LPS) stimulation, without affecting human leucocyte antigen D-related and CD80 MFI. In the same co-culture model, DCs produced increased amounts of interleukin (IL)-10 when co-cultured with ECP-treated lymphocytes and stimulated with LPS, while IL-12 and tumour necrosis factor-alpha production were not affected. These results suggest that reinfusion of large numbers of autologous apoptotic lymphocytes is significant for the therapeutic outcome of ECP through down-regulation of co-stimulatory molecules on DCs, inducing non-fully mature DCs with a low signal 2 and up-regulation of IL-10, which is an immunosuppressive cytokine.

Through physiologic interactions with its ligands CD58 (lymphocyte function-associated Ag-3, LFA-3) and CD59, the T cell glycoprotein CD2 plays a role in T cell signaling and promotes lymphocyte adhesion. We have recently demonstrated that the interaction of CD2 with CD58 is dynamic: TCR stimulation or treatment with the phorbol ester PMA rapidly up-regulates CD2 ligand avidity, and this regulation requires the carboxyl-terminal asparagine residue of the CD2 cytoplasmic domain. Here we have analyzed the regulation of CD2 avidity for CD58, as assessed by the binding of CD2+ cells to purified CD58 and by the formation of rosettes between CD2+ cells and SRBC. In murine T cell hybridomas transfected with human CD2, we show that, unlike CD2-mediated IL-2 production, cell surface expression of the TCR-CD3 structure is not required for up-regulation of CD2 ligand avidity. TCR-initiated up-regulation of CD2 avidity requires the activity of both protein tyrosine kinases and protein kinase C. Agents which elevate intracellular levels of cAMP also up-regulate CD2 ligand avidity and act either distal to or independently of protein kinase C and protein tyrosine kinases. Cell lines expressing single amino acid substitutions of the carboxyl-terminal asparagine of CD2 are incapable of avidity regulation by TCR signaling, PMA treatment, or elevation of intracellular cAMP levels, demonstrating that each of these stimuli utilizes a common structural element for regulating CD2 avidity. The response to both cAMP and phorbol ester treatment distinguishes the regulation of CD2 avidity from that of a second major adhesion pathway, LFA-1 (CD11a/CD18)/ICAM-1 (CD54) and from that of the TCR coreceptor CD8. These observations identify the signaling events involved in the regulation of CD2 avidity and help to define the signal transduction processes that participate in "inside-out" signaling.

Possible effects of multi-wall carbon nanotubes (MWCNTs) on immune and inflammatory responses were examined in mice. Female ICR mice were given a single intraperitoneal administration (2 mg/kg body weight) of either MWCNTs, carbon black (CB), or crocidolite (blue asbestos) and controls received a vehicle of 2% sodium carboxymethyl cellulose (CMC Na). In the peritoneal cavity of MWCNT-administered mice, the liver had changed to a rounded shape and fibrous adhesions were seen on internal organs. Peritoneal cells overexpressed mRNA for genes of T helper (Th)2 cytokines (interleukin [IL]-4, IL-5, and IL-13), Th17 cytokine (IL-17), pro-inflammatory cytokines/chemokines (IL-1β, IL-33, tumor necrosis factor α, and monocyte chemotactic protein-1), and myeloid differentiation factor 88 for at least 2 weeks after the administration of MWCNTs, while those of Th1 cytokine genes (IL-2 and interferon γ) were overexpressed several weeks later and expression levels remained high up to 20 weeks. In MWCNT-treated mice, the numbers of leukocytes, monocytes, and granulocytes in the peripheral blood and the expression of the leukocyte adhesion molecules, cluster of differentiation (CD)49d and CD54, on granulocytes were increased 1 week after administration and remained high up to week 20. Production of ovalbumin-specific IgM and IgG(1) was enhanced by MWCNTs. These changes were not observed after CB or crocidolite administration. Thus, this study showed that MWCNTs exhibited sustained stimulating effects on immune and inflammatory responses, unlike the other mineral fibers with structural similarities.

Distinct subsets of dendritic cells (DCs) based on the origin, phenotypes, and the nature of the signals that promote DC maturation can determine polarized immune responses of T cells. In this study, DCs were cultured from mouse bone marrow (BM) progenitors in granulocyte-macrophage colony-stimulating factor (GM-CSF). To generate mature DCs (mDCs), lipopolysaccharide (LPS) was used in the culture for 24 h. LPS-stimulated DCs were phenotypically mature, which exhibited strongly upregulated CD40, B7.1, and B7.2 compared to non-LPS-stimulated immature DCs (imDCs). Both mDCs and imDCs expressed high levels of MHC class II but low level of CD54. mDCs produced higher levels of IL-10 and lower IL-12 compared to imDCs. No IFN-gamma or IL-4 was found in both groups. When mDCs were injected intraperitoneally (i.p.) to the mice with experimental autoimmune encephalomyelitis (EAE), the severity of clinical signs and inflammation in the CNS was significantly suppressed compared to imDC-injected mice (p<0.01) and PBS-injected mice (p<0.02). Moreover, lymphocytes from mDC-injected mice produced lower level of IL-12, IFN-gamma, but higher level of IL-10, compared to imDC-injected and non-DC-injected mice. We conclude that BM-mDCs, but not BM-imDCs, promote Th2 differentiation and have the potential for suppression of inflammatory demyelination.

Mice were exposed to pure oxygen for various times to explore the pulmonary platelet trapping associated with alveolar damage, its mechanism, and its role in the lesions. Platelet sequestration, evaluated by electron microscopy and by injection of radiolabeled platelets, was detectable after 72 h and reached a maximum after 96 h of exposure (i.e., shortly before death). Circulating platelets (analyzed by Facscan) showed some increase in the expression of CD11a and CD62, but little change in CD31 and CD61. Both platelet activation and lung sequestration were dependent on TNF-alpha, since antibody against TNF-alpha reduced the expression of CD11a on circulating platelets and their sequestration in the lung. Lung platelet sequestration was also decreased by anti-CD11a MoAb. Northern blot analysis of lung mRNA isolated at 96 h of oxygen exposure revealed a 7-fold increase in CD54 (intercellular adhesion molecule-1 [ICAM-1]) and a 2.5-fold increase in TNF-alpha mRNAs respectively. These results demonstrate that the platelet pulmonary trapping induced by hyperoxia is dependent upon TNF-alpha and the CD11a-CD54 adhesion molecules. However, platelet trapping does not appear to play an important pathogenic role in acute oxygen injury, since treatments that decrease trapping (anti-TNF-alpha, anti-CD11a, or antibody-induced thrombocytopenia) did not markedly attenuate the alveolar damage.

Infection of susceptible mice with Plasmodium berghei Anka leads to a syndrome of severe or cerebral malaria. Tumor necrosis factor (TNF) contributes to this syndrome, apparently by acting on its receptor 2 (TNFR2) because TNFR1-/- are susceptible, whereas TNFR2-/- mice are resistant. In this work, we confirmed the essential role of the TNFR2 in cerebral malaria because 6 to 8 days after Plasmodium berghei Anka infection, hypothermia, coma, and death were observed in +/+ or TNFR1-/-, but never in TNFR2-/-, mice. TNF production, evaluated by the serum levels or the mRNA levels in the brain, spleen or lung, was similar in +/+, TNFR1-/-, or TNFR2-/- mice. Macrophage or parasitized red blood cell sequestration in brain or lung was similar in TNFR1-/- and TNFR2-/- mice. Accordingly, up-regulation of CD54 or CD40 in brain or lung was also similar in TNFR1-/- or TNFR2-/- mice. Platelet loss, manifested by thrombocytopenia and the presence of microparticles in plasma, was similar in TNFR1-/- or TNFR2-/- mice. Breakdown of the blood-brain barrier, detected by the diffusion of tracers, was attenuated in both TNFR1-/- and TNFR2-/-, compared with +/+, mice. Endothelial cells from brain capillaries, examined by transmission electron microscopy, were similar in infected TNFR1-/- or TNFR2-/- mice, whereas the basement membrane was enlarged in TNFR1-/- mice. Hypothermic mice were also hyperglycemic, and this was evident in +/+ and TNFR1-/-, but not in TNFR2-/-, mice. In addition, infected +/+ and TNFR1-/- mice became insulin resistant, while in contrast TNFR2-/- became extremely insulin sensitive. This study supports the possibility that coma and death are mediated not by cell sequestration or breakdown of vascular permeability, similar in TNFR1-/- or TNFR2-/- mice, but by metabolic disturbances selectively mediated by the TNFR2.

We have optimized assays to measure mitogen-stimulated rat lymphocyte activation in whole blood and have used these assays to quantitate the potencies of immunosuppressive drugs with different mechanisms of action. To define the optimal conditions for measuring T cell functions in whole blood, the effects of different concentrations of mitogens that activate T cells through calcium-dependent and -independent pathways were measured over time. Proliferation was measured by tritium-labeled thymidine ([3H]-TdR) incorporation and by flow cytometric analysis of proliferating cell nuclear antigen (PCNA)/DNA content. Furthermore, we detected the increases in percent expression of cell-surface activation antigens (CD25, CD134, CD71, CD11a and CD54). Concanavalin A (Con A) stimulated maximum lymphocyte proliferation and expression of T cell surface activations by 72-96 h, which was 48 h later than stimulation by phorbol 12-myristate 13-acetate (PMA) plus anti-CD28 monoclonal antibody (mAb) or PMA plus ionomycin (IONO). Addition of sirolimus, tacrolimus, cyclosporine or the active metabolite of leflunomide, A77 1726, to mitogen-stimulated whole blood produced drug concentration-dependent inhibitions of lymphocyte proliferation and expression of cell surface activation antigen expression. From these data, we determined drug potencies (inhibitory concentration of 50%, IC(50)) and drug concentrations causing maximum inhibition of T cell functions (I(max)). We developed simple and reproducible assays to measure different lymphocyte functions in whole blood cultures. These assays were used to investigate the mechanisms of different immunosuppressive drugs. These methods can be exploited to measure T cell functions in blood collected from subjects treated with immunosuppressants in vivo.

Increased mechanical stretch of alveolar type II (ATII) cells occurs during mechanical ventilation. The effects of three patterns of stretching rat ATII cells (frequency [min-1]-Deltasurface area [%]: S40-13, S60-13, S40-30) were compared with those in static cultures at 12, 18, and 24 h. Cell viability and expression of cyclooxygenase-2,5-lipoxygenase, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) were characterized. Supernatants were analyzed for eicosanoids, nitrite, cytokines, and stimulatory effects on rat lymphocytes. S40-13 simulates normal breathing; the other patterns increased amplitude and frequency. There were no significant differences between S40-13 and static cultures. S60-13 only significantly increased the supernatant nitrite (11.2+/-1.6 versus 3.9+/-0.4 microM at 24 h). S40-30 significantly reduced the number of trypan blue-excluding cells, increased the supernatant concentration of TXB2 (4.1+/-0.61 versus 2.2+/-0.36 pg/ml), 6-keto-PGF1alpha (8.7+/-1.0 versus 6.7+/-0.52 pg/ml), cysteinyl-LT (12.2+/-2.0 versus 6.1+/-0.75 pg/ml) and nitrite (7.2+/-1.7 versus 3.9+/-0.4 microM). S40-30 did not alter the release of tumor necrosis factor-alpha and monocyte chemotactic protein-1, but significantly reduced the concentration of the anti-inflammatory interleukin-10 (20.8+/-13.3 versus 130+/-21.5 pg/ml). Expression of cyclooxygenase-2/5-lipoxygenase was increased/decreased; expression of iNOS/eNOS was unchanged by high-amplitude stretch. Supernatants from S40-30 experiments caused lymphocyte activation measured by CD71 and CD54 surface expression. Continuing mechanical distension of ATII cells contributes to an inflammatory response by a shift in the balance of pro- and anti-inflammatory mediators.

In rheumatoid arthritis (RA) synovial fibroblasts are activated by growth factors and cytokines to proliferate and to express matrix-degrading proteases and pro-inflammatory cytokines. This contributes to cartilage degradation and joint destruction. To analyse the parameters that lead to activation of synovial fibroblasts, we established a stable human synoviocyte line (K4IM) from a healthy donor by immortalization with SV40 T antigen (TAg). Characterizing the phenotype of the immortalized K4IM cells, we found that they maintained CD44, CD54 (intercellular adhesion molecule; ICAM-1) and CD95 (Fas) expression, but lost the expression of CD106 (vascular cell adhesion molecule 1; VCAM-1) and the receptors for interleukin 1 (IL-1) and platelet-derived growth factor (PDGF). We also monitored normal expression kinetics of transcription factor Egr-1 upon activation with tumor necrosis factor alpha (TNF-alpha) or synovial fluid from RA patients. In addition, we showed that HLA-DR expression could still be upregulated by recombinant interferon gamma (rINF-gamma). The immortalized K4IM cell line therefore represents a valuable and unique tool to study mechanisms that induce or maintain synoviocyte activation.

Dynamic regulatory mechanisms prevent autoreactive T cell activation. Upon T cell receptor crosslinking, CD4+CD25+ T regulatory (T(R)) cells block both the proliferation and cytokine production of CD4+CD25- effector cells in an apparent antigen non-specific manner. Within the T(R)population, L-selectin (CD62L)(hi)T(R)cells have been described as more efficient suppressors of T cell proliferation than CD62L(low)T(R)cells. We have previously reported that CD4+CD25+CD62L(hi)T(R)cells express elevated levels of two additional adhesion molecules, ICAM-1 (CD54) and P-selectin (CD62P) in comparison to non-T(R)cells. In the current study, we investigated the functional contribution of CD54 and CD62P expression to the suppressive phenotype of T(R)cells both in vitro and in vivo. While the CD4+CD25+ T(R)cell population was demonstrated to be significantly larger in CD62P-/- mice than in wild-type C57BL/6 mice, CD62P-/- T(R)cell function was deficient in vitro, but not in vivo. Interestingly, we detected no deficiencies in T(R)cell numbers or effector function in CD54-/- mice suggesting that T(R)cells may differ from effector CD4+ T cells in the requirement for CD54 expression within the immunological synapse. Collectively, these findings indicate that CD62P may influence T(R)cell differentiation/development and that T(R)cell activation occurs independently of CD54 expression.

OBJECTIVE

To study the effects of different disease-modifying antirheumatic drugs (DMARD) on different events mediated by IL-15-activated lymphocytes.

METHODS

Peripheral blood lymphocytes (PBL) were isolated from healthy donors and activated with IL-15 after exposure to different DMARD: leflunomide, cyclosporin A, methotrexate, mycophenolic acid, FK-506, sulphasalazine and sodium aurothiomalate. The expression of different surface molecules on the PBL was then determined by flow cytometry. Cells were also co-cultured with the monocytic cell line THP-1 and the tumour necrosis factor (TNF) concentration in the supernatant was measured after 24 h using an immunoenzyme assay. The effect of the aforementioned drugs on IL-17 production by IL-15-activated PBL was also studied.

RESULTS

Treatment of PBL with leflunomide, cyclosporin A and FK-506 inhibited the IL-15-induced expression of both CD54 and CD69 by PBL, as well as TNF production in co-cultures of activated PBL and THP-1 cells. The downregulation of CD54 and CD69 in PBL was correlated with the inhibition of TNF production. Likewise, leflunomide, cyclosporin A and FK-506 all inhibited IL-17 production in IL-15-activated PBL. Interestingly, the effect of leflunomide was not reverted by the presence of uridine in the medium. In addition, leflunomide inhibited the phosphorylation of STAT6 in vitro.

CONCLUSIONS

Inhibition of the JAK/STAT pathway may represent an additional effect of leflunomide in chronic polyarthritis because it impairs certain events that control proinflammatory TNF and IL-17 cytokine production.

Metastatic colorectal cancer is usually progressive despite infiltration of the tumours by T lymphocytes, suggesting that these tumour-infiltrating lymphocytes (TILs) are functionally deficient. Recently, TILs from other tumours have been shown to express reduced levels of the T-cell receptor signal-transducing CD3-zeta chain. We were interested to determine whether a similar abnormality existed in TILs from human colorectal hepatic metastasis (CHM) and, if so, whether correcting the abnormality in vitro would restore anti-tumour activity and provide support for the development of immunotherapy for colorectal hepatic metastases. Twelve of 19 TILs from colorectal hepatic metastases were successfully expanded in vitro in high-dose recombinant interleukin 2 (rlL-2) and their specific anti-tumour cytolytic activity was determined. CD3-positive (CD3+) TILs were HLA-Drhigh and CD69high, suggesting that they had been activated by exposure to antigen but expressed low levels of CD25, CD71 and the nuclear proliferation antigen Ki-67. Furthermore, they showed reduced expression of CD3-zeta compared with autologous peripheral blood T cells (PBTs) and failed to proliferate in the absence of high-dose rIL-2. Expansion of TILs in rIL-2 resulted in restoration of CD3-zeta expression and the ability to lyse K562 and Daudi cells but not autologous tumour cells. The absence of autologous tumour-specific cytolytic T-cell (CTL) activity may be due to the poor immunogenicity of colorectal tumour cells, which we found expressed only low levels of MHC I antigens and CD54 and failed to express MHC II antigens or the co-stimulatory molecules CD80, CD86 or CD106. The inability of rIL-2 to generate tumour-specific CTLs despite restoration of CD3-zeta expression and the presence of an intact lytic mechanism suggests that successful immunotherapy may require the development of strategies to increase the immunogenicity of this tumour.

We analyzed the accessory function of malignant B cells from non-Hodgkin's lymphomas (NHLs). Among the 70 samples of malignant B cells included, four patterns of expression of the costimulatory molecules CD80 and CD86 were distinguished (+/+, +/-, -/+ and -/-). In two-thirds of the cases, CD80, CD86, or both were expressed. To investigate the relevance of these molecules for tumor immunogenicity, mixed lymphocyte reactions (MLR) were performed with allogeneic responding T cells and malignant B cells from nine NHL patients. Regardless of the level of expression of CD80 and CD86, significant proliferation was induced in the responder cells. The addition of monoclonal antibodies directed against CD80 and CD86 at the beginning of MLR almost completely inhibited this proliferation. We show that, during MLR, a high level of expression of CD80 and CD86 was induced in NHL B cells. Thus, cooperation between responding and stimulator cells seems to occur during MLR, allowing induction of optimal accessory function of B cells. We investigated whether malignant B cells cultured with CD40-L-transfected L cells in the presence of IL-4 could augment their antigen-presenting cell (APC) functions. The culture of NHL B cells in this sytem induced strong upregulation of the expression of CD80 and CD86 as well as other molecules involved in accessory cell functions (HLA class I, CD54, and CD58). In half of the cases, this activation resulted in enhanced proliferation of allo-T cells as compared to the proliferation induced by nonactivated malignant B cells. Our results show that NHL B cells are able to express functional CD80 and CD86 and to be fully competent APC. This suggests that the absence of an efficient T cell-mediated antitumor response in vivo is not related to a deficiency in the APC functions of malignant B cells.

Dendritic cell (DC)-tumor fusion hybrid vaccine which facilitates antigen presentation represents a new powerful strategy in cancer therapy. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between wild-type J558 or engineered J558-IL-4 myeloma cells secreting cytokine interleukin-4 (IL-4) and immature DCs (DC(IMAT)) or relative mature DCs (DC(RMAT)). DC(RMAT) displayed an up-regulated expression of immune molecules (Ia(d), CD40, CD54, CD80 and CD86) and certain cytokines/chemokines, and enhanced ability of allogeneic T cell stimulation when compared to DC(IMAT). These DCs were fused with myeloma cells by polyethylene glycol (PEG). The fusion efficiency was approximately 20%. Our data showed that immunization of C57BL/6 mice with DC(RMAT)/J558 hybrids induced protective immunity against a high dose of J558 tumor challenge (1x10(6) cells) in 3 out of 10 immunized mice, compared with no protection seen in mice immunized with DC(IMAT)/J558 hybrids. Furthermore, immunization of mice with engineered DC(RMAT)/J558-IL-4 hybrids elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro and induced more efficient protective immunity (10/10 mice; tumor free) against J558 tumor challenge in vivo than DC(RMAT)/J558 hybrid vaccines. The results demonstrate the importance of DC maturation in DC-tumor hybrid vaccines and indicate that the engineered fusion hybrid vaccines which combine gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy.

Intercellular adhesion molecule-1 (ICAM-1; CD54) is an adhesion molecule constitutively expressed in abundance on the cell surface of type I alveolar epithelial cells (AEC) in the normal lung and is a critical participant in pulmonary innate immunity. At many sites, ICAM-1 is shed from the cell surface as a soluble molecule (sICAM-1). Limited information is available regarding the presence, source, or significance of sICAM-1 in the alveolar lining fluid of normal or injured lungs. We found sICAM-1 in the bronchoalveolar lavage (BAL) fluid of normal mice (386 +/- 50 ng/ml). Additionally, sICAM-1 was spontaneously released by murine AEC in primary culture as type II cells spread and assumed characteristics of type I cells. Shedding of sICAM-1 increased significantly at later points in culture (5-7 days) compared with earlier time points (3-5 days). In contrast, treatment of AEC with inflammatory cytokines had limited effect on sICAM-1 shedding. BAL sICAM-1 was evaluated in in vivo models of acute lung injury. In hyperoxic lung injury, a reversible process with a major component of leak across the alveolar wall, BAL fluid sICAM-1 only increased in parallel with increased alveolar protein. However, in lung injury due to FITC, there were increased levels of sICAM-1 in BAL that were independent of changes in BAL total protein concentration. We speculate that after lung injury, changes in sICAM-1 in BAL fluid are associated with progressive injury and may be a reflection of type I cell differentiation during reepithelialization of the injured lung.

The role of platelets was investigated in two models of lipopolysaccharide (LPS)-induced toxicity in mice: the systemic reaction, provoked by intravenous LPS injection in D-galactosamine-sensitized recipients, which results in host death, and the local reaction, elicited in the skin by sequential injections of LPS and tumor necrosis factor alpha at 24-h intervals, which results in hemorrhagic necrosis. In both models, the depletion of platelets with a rabbit polyclonal or a mouse monoclonal antiplatelet immunoglobulin G afforded significant protection. In the local reaction, studies of the distribution of 111In-labelled platelets as well as optical and electron microscopy showed that platelets are localized in the dermal venules before hemorrhage occurs. Anti-CD11a (LFA-1) and anti-CD54 (ICAM-1) monoclonal antibodies prevented both platelet localization and hemorrhagic necrosis, and these determinants were detected on mouse platelets by immunofluorescence. The antiplatelet monoclonal antibody did not reduce the localization of polymorphonuclear leukocytes in the dermal venules, as shown by histological sections. Thus, in the local reaction, the stimulation with LPS and tumor necrosis factor alpha leads to a binding of platelets to the endothelium of venules by their beta 2 integrins, which seems necessary for the development of the hemorrhagic necrosis.

The immune responsiveness of women is altered during pregnancy in order to retain protective properties against disease and at the same time allow tolerance of the fetus. Diseases such as pre-eclampsia (PE) have been suggested to arise as a result of maladaptations in these immune alterations. Here we evaluate the effect of PE on the composition of peripheral blood lymphocyte subpopulations using lymphocyte surface antigen expression. Fifty-four women of various parities with pregnancy-induced hypertension (PIH) (39 non-proteinuric and 14 proteinuric) and matched controls (30 normotensive pregnant women (NTP) and 15 healthy non-pregnant women (NP)) were investigated. Monoclonal antibodies specific for human T lymphocytes and subpopulations: CD2, CD3, CD4, CD8, CD19 and activation markers: CD25, CD45RA, CD45RO, CD54 AND HLA(-)DR were used and detected using a two-colour fluorescence analysis with an automated flow cytometer. The total number of T lymphocytes: CD2, CD3, CD4, CD8 and CD19 were significantly decreased in PIH particularly PE (P<0.05). T cells expressing NK surface markers (CD3/CD16(+)CD56) and CD4 cells expressing HLA(-)DR were higher in PE. CD8(+)HLA(-)DR(+) cells and T-helper cells expressing adhesion molecules) CD4(+)CD54(+)) were higher in NTP than in NP and PE (P<0.05, 0.05). PE is associated with elevated levels of CD4(+)HLA(-)DR(+), and CD3(+)NK cells but decreased total numbers of T lymphocytes, and the CD3(+)CD25(+) subpopulation. These findings indicate systemic alterations in maternal immunity associated with the PE state. This feature of the disease may contribute to abnormal adaptation to pregnancy resulting in PE and PIH, promoting adverse outcomes including pregnancy loss.

Recent studies of rat anti-glomerular basement membrane (anti-GBM) disease have demonstrated a functional role for ICAM-1 in the entry of leukocytes into the glomerulus, both in the early polymorphonuclear (PMNL) influx and the more delayed monocyte/macrophage infiltration. In the current study we used immunogold ultrastructural techniques to identify the exact sites of expression of ICAM-1 (CD54) in the glomerulus and the expression of CD11a and CD18 by infiltrating glomerular leukocytes in the first 24 hours of accelerated anti-GBM disease in rats. In normal rats there was constitutive ICAM-1 expression on the luminal surface of the glomerular endothelium and parietal epithelium of Bowman's capsule. In disease ICAM-1 expression was progressively increased over 24 hours on a thickened, reactive glomerular endothelium, being most prominent on endothelium adjacent to the mesangial stalks. Mesangial cells demonstrated surface ICAM-1 expression only in focal areas of superficial mesangiolysis. PMNL, the predominant glomerular inflammatory cell in the first 12 hours of accelerated anti-GBM GN, expressed abundant surface CD18 which was present at the sites of adhesion of the PMNL to the glomerular endothelium. In contrast PMNL expressed only very sparse surface CD11a, suggesting that another beta 2 integrin, Mac-1, which shares a common beta chain with LFA-1 may be the more important PMNL counter receptor for ICAM-1 in the glomerulus. Glomerular monocyte/macrophage infiltration became evident within glomerular capillary loops and the mesangium from 6 to 24 hours. These adherent and migrating leukocytes expressed abundant surface CD11a and moderate CD18 particularly at their sites of adhesion to glomerular endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer stem cells (CSCs) are thought as the source of tumor maintaining and many CSCs markers have been identified. Regarding the heterogeneity in gastric cancer (GC), TNM stage is not enough to accurately predict the prognosis. The aim of this study was to investigate the clinical significance of CSCs markers (Lgr5, Oct4, CD133, EpCAM, CD54 and Sox2) and establish a new model based on these markers to accurately predict prognosis of GC. We retrospectively enrolled 377 GC tissues from January 2006 to October 2012 to perform immunohistochemistry (IHC), and 93 pairs of GC tissues and corresponding adjacent normal gastric tissues to perform quantitative PCR (qPCR) from December 2011 to October 2012. The clinicopathological and follow-up characteristics were collected. In IHC, Oct4, CD133 and EpCAM were independently related to tumor progression, while Sox2 were associated with well or moderate differentiation (all p<0.05). Cox regression showed that Oct4-EpCAM was an independently prognostic factor, indicating that double low expression of Oct4-EpCAM group had significantly better prognosis than control group (p=0.035). Regarding qPCR, CD133 was an independent prognostic factor, showing that the prognosis of patients with CD133 high expression was significantly worse than that of patients with CD133 low expression (p<0.001). The prognostic prediction accuracy of nomogram based on Oct4-EpCAM expression in IHC was significantly better than TNM stage alone (p=0.003). Low expressions of Oct4-EpCAM in IHC and CD133 in qPCR were favorable prognostic factors in GC. The nomogram based on Oct4-EpCAM was valuable in prognostic prediction of GC patients.