Enhancement of antitumor radiation efficacy and consistent induction of the abscopal effect in mice by ECI301, an active variant of macrophage inflammatory protein-1alpha.

PURPOSE: We studied whether i.v. administration of a chemokine after local tumor site irradiation could prevent remaining, as well as distant, nonirradiated tumor cell growth by leukocyte recruitment. EXPERIMENTAL DESIGN: Tumors were implanted s.c. in the right or both flanks. After local irradiation at the right flank, ECI301, a human macrophage inflammatory protein-1alpha variant was injected i.v. Tumor volumes were measured every 3 days after treatment. RESULTS: In Colon26 adenocarcinoma-bearing BALB/c mice, repeated daily administration (over 3-5 consecutive days) of 2 mug per mouse ECI301 after local irradiation of 6 Gy prolonged survival without significant toxicity, and in about half of the treated mice, the tumor was completely eradicated. Three weekly administrations of ECI301 after local irradiation also led to significant, although less effective, antitumor radiation efficacy. ECI301 also inhibited growth of other syngenic tumor grafts, including MethA fibrosarcoma (BALB/c) and Lewis lung carcinoma (C57BL/6). Importantly, tumor growth at the nonirradiated site was inhibited, indicating that ECI301 potentiated the abscopal effect of radiation. This abscopal effect observed in BALB/c and C57BL/6 mice was tumor-type independent. Leukocyte depletion studies suggest that CD8+ and CD4+ lymphocytes and NK1.1 cells were involved. CONCLUSIONS: Marked inhibition of tumor growth at the irradiated site, with complete tumor eradication and consistent induction of the abscopal effect, was potentiated by i.v. administration of ECI301. The results of this study may offer a new concept for cancer therapy, namely chemokine administration after local irradiation, leading to development of novel therapeutics for the treatment of advanced metastatic cancer.

Maternal microchimerism in biliary atresia.

BACKGROUND: The aim of this study was to determine the existence and extent of maternal microchimerism in the livers of biliary atresia (BA) patients. METHODS: Two series of investigations were performed based on the sex of our subjects. Subjects for series I were men, of which 6 had BA. Livers were analyzed using X and Y chromosome probes and fluorescent in situ hybridization. Subjects for series II were woman. Nine BA cases and their mothers were HLA typed (class I). Daughter livers were also tested for antibodies to maternal and other HLA. Two cases of neonatal hepatitis, 2 cases of Alagille syndrome, and 1 case of Byler syndrome acted as controls. RESULTS: All male BA livers were found to contain a mixture of cells with 1 and 2 X chromosomes (ie, XY or XX). All livers from male controls had only 1 X chromosome (ie, XY). All female BA subjects had varying intensities of antimaternal HLA class I (HLA-A) antibodies in their bile duct epithelium and hepatocytes (strong, 5; mild, 3; weak, 1). The liver from the female control did not display any antimaternal HLA class I antibodies (HLA-Ab). CONCLUSION: Our preliminary data appear to show that maternal microchimerism is present within the livers of patients with progressive postnatal type BA. We suggest that BA could in fact be a graft-vs-host disease masquerading as an autoimmune reaction triggered by maternal microchimerism, and we intend to pursue this hypothesis further to clarify the etiology of BA.

Quantitative analysis of eosinophil chemotaxis tracked using a novel optical device -- TAXIScan.

We have reported previously the development of an optically accessible, horizontal chemotaxis apparatus, in which migration of cells in the channel from a start line can be traced with time-lapse intervals using a CCD camera (JIM 282, 1-11, 2003). To obtain statistical data of migrating cells, we have developed quantitative methods to calculate various parameters in the process of chemotaxis, employing human eosinophil and CXCL12 as a model cell and a model chemoattractant, respectively. Median values of velocity and directionality of each cell within an experimental period could be calculated from the migratory pathway data obtained from time-lapse images and the data were expressed as Velocity-Directionality (VD) plot. This plot is useful for quantitatively analyzing multiple migrating cells exposed to a certain chemoattractant, and can distinguish chemotaxis from random migration. Moreover precise observation of cell migration revealed that each cell had a different lag period before starting chemotaxis, indicating variation in cell sensitivity to the chemoattractant. Thus lag time of each cell before migration, and time course of increment of the migrating cell ratio at the early stages could be calculated. We also graphed decrement of still moving cell ratio at the later stages by calculating the duration time of cell migration of each cell. These graphs could distinguish different motion patterns of chemotaxis of eosinophils, in response to a range of chemoattractants; PGD(2), fMLP, CCL3, CCL5 and CXCL12. Finally, we compared parameters of eosinophils from normal volunteers, allergy patients and asthma patients and found significant difference in response to PGD(2). The quantitative methods described here could be applicable to image data obtained with any combination of cells and chemoattractants and useful not only for basic studies of chemotaxis but also for diagnosis and for drug screening.

The role of monocyte chemoattractant protein-1 in biliary atresia.

BACKGROUND: The aim of this study was to explain the role of monocyte chemoattractant protein-1 (MCP-1) in biliary atresia (BA). METHODS: Concentrations of serum MCP-1 and collagen type IV were measured in 38 patients with BA by using commercially available kits. MCP-1 was also assessed in liver biopsy specimens by using immunohistochemistry. Subjects were classified into groups. Group 1 comprised BA patients with normal liver function (n = 13), group II comprised BA patients with moderate liver dysfunction (n = 18), group III comprised BA patients older than 20 years awaiting liver transplantation (n = 7), and the control group comprised age-matched patients without evidence of liver disease (n = 23). RESULTS: Serum MCP-1 levels were significantly increased in group II compared with group I (P < .0001) and the control group (P < .0001). Serum MCP-1 levels in group III were lower than in the control group (P < .0001). There was a significant linear correlation between serum MCP-1 levels and type IV collagen levels in group II. Group II subjects with portal hypertension (PH) had higher MCP-1 levels than those without PH (P = .0009). Biopsy specimens showed MCP-1 was expressed mainly on biliary epithelial cells, vascular endothelial cells, and hepatocytes in group II. CONCLUSIONS: These findings suggest that MCP-1 probably plays a significant role in the development of progressive liver fibrosis in BA.

High expression of eosinophil chemoattractant ecalectin/galectin-9 in drug-induced liver injury.

BACKGROUND: Ecalectin/galectin-9 (ECL/GL9) is an eosinophil chemoattractant isolated from T lymphocytes. Drug-induced liver injury (DILI), often caused by an allergic mechanism, is occasionally accompanied by eosinophilic infiltration. In this study, we intended to determine whether DILI can induce augmentation of ECL/GL9 expression. Further, we investigated whether this augmentation is associated with tissue eosinophilia. METHODS: We examined the expression of ECL/GL9 in biopsy specimens of DILI using the immunohistochemical technique. A rabbit anti-ECL/GL9 antibody was produced by immunizing rabbits with synthetic peptide corresponding to a molecular epitope of ECL/GL9. Thereafter, immunohistochemical staining with the use of this antibody was performed on 16 DILI needle biopsy specimens, and on biopsy specimens of chronic viral hepatitis, liver cirrhosis, and normal liver tissues as controls. RESULTS: In all cases of DILI specimens, but not in control liver specimens, a clear positive staining for ECL/GL9 was observed. Such positive staining was noted on Kupffer cells, fibroblasts, and histiocytes, but not on lymphocytes or hepatocytes. However, the intensity of immunolabeling did not correlate with the extent of eosinophile leukocyte infiltration. CONCLUSION: High expression of ECL/GL9 is suggested to be a specific finding of DILI. However, tissue eosinophilia in DILI cannot be explained by the augmentation of ECL/GL9 expression.

Various adhesion molecules impair microvascular leukocyte kinetics in ventilator-induced lung injury.

Although the endothelial expression of various adhesion molecules substantially differs between pulmonary microvessels, their importance for neutrophil and lymphocyte sequestration in ventilator-induced lung injury (VILI) has not been systematically analyzed. We investigated the kinetics of polymorphonuclear cells (PMN) and mononuclear cells (MN) in the acinar microcirculation of the isolated rat lung with VILI by real-time confocal laser fluorescence microscopy, with or without inhibition of ICAM-1, VCAM-1, or P-selectin by monoclonal antibodies (MAb). Adhesion molecules in each microvessel were estimated by intravital fluorescence microscopy or immunohistochemical staining. In high tidal volume-ventilated lungs, 1) ICAM-1, VCAM-1, and P-selectin were differently upregulated in venules, arterioles, and capillaries; 2) venular PMN rolling was improved by inhibition of ICAM-1, VCAM-1, or P-selectin, whereas arteriolar PMN rolling was improved by ICAM-1 or VCAM-1 inhibition; 3) capillary PMN entrapment was ameliorated only by anti-ICAM-1 MAb; and 4) MN rolling in venules and arterioles and MN entrapment in capillaries were improved by ICAM-1 and VCAM-1 inhibition. In conclusion, the contribution of endothelial adhesion molecules to abnormal leukocyte behavior in VILI-injured microcirculation is microvessel and leukocyte specific. ICAM-1- and VCAM-1-dependent, but P-selectin-independent, arteriolar PMN rolling, which is expected to reflect the initial stage of tissue injury, should be taken as a phenomenon unique to ventilator-associated lung injury.

Stem cell factor has a suppressive activity to IgE-mediated chemotaxis of mast cells.

Stem cell factor (SCF), which is well known as a cytokine capable of amplifying development and functions of mast cells, is mainly released from fibroblasts in the peripheral tissue. To investigate whether SCF controlled chemotactic migration of mast cells induced by IgE-specific Ag, murine bone marrow-derived cultured mast cells (BMCMC) and human cord blood-derived cultured mast cells (HuCMC) were preincubated with SCF. Although BMCMC and HuCMC sensitized with IgE directly moved toward specific Ag, preincubation for even 1 h with an optimal dose of SCF suppressed the IgE-mediated chemotactic movement. No or little inhibitory effect of SCF was detected in BMCMC derived from c-kit receptor-defect WBB6F1-W/Wv mice. In contrast, preincubation of BMCMC and HuCMC with SCF enhanced beta-hexosaminidase release and Ca2+ mobilization in response to Ag after sensitization with IgE. Using the real-time record of chemotactic migration, BMCMC preincubated with SCF manifested motionless without degranulation. These results suggest that locally produced SCF may have an inhibitory effect on chemotaxis of mast cells, contributing to their accumulation and enhancement of functions at the peripheral site in allergic and nonallergic conditions.

A novel optical assay system for the quantitative measurement of chemotaxis.

We have developed an optically accessible, horizontal chemotaxis apparatus consisting of an etched silicon substrate and a flat glass plate, both of which form two compartments with a 5-microm-deep microchannel in between. The device is held together with a stainless steel holder with holes for injecting cells and a chemoattractant to the different compartments. Migration of cells in the channel is traced with time-lapse intervals using a CCD camera. By developing a method for aligning cells at the edge of the channel, we could successfully reduce the number of cells required for a chemotactic assay, depending on the experiment, to 100 or less. To prevent ceaseless flow of contents between the adjacent compartments via the communicating microchannel, a space at the top end of the holder was filled with medium after aligning the cells. By using a fluorescent probe, we demonstrated experimentally that a stable concentration gradient could be maintained. Furthermore, we determined theoretical details of the gradient established using a model chemokine and a computational fluid dynamics code. Reproducible kinetic results of cell migration were obtained using human neutrophils and IL-8 as a model. Migration of other cells such as eosinophils, basophils and Jurkat lymphocytes toward the appropriate chemokines were also demonstrated.

Distinct sulfation requirements of selectins disclosed using cells that support rolling mediated by all three selectins under shear flow. L-selectin prefers carbohydrate 6-sulfation totyrosine sulfation, whereas p-selectin does not.

l- and P-selectin are known to require sulfation in their ligand molecules. We investigated the significance of carbohydrate 6-sulfation and tyrosine sulfation in selectin-mediated cell adhesion. COS-7 cells were genetically engineered to express P-selectin glycoprotein ligand-1 (PSGL-1) or its mutant in various combinations with 6-O-sulfotransferase (6-Sul-T) and/or alpha1-->3fucosyltransferase VII (Fuc-T VII). The cells transfected with PSGL-1, 6-Sul-T, and Fuc-T VII cDNAs supported rolling mediated by all three selectins and provided the best experimental system so far to estimate kinetic parameters in selectin-mediated cell adhesion for all three selectins using the identical rolling substrate and to compare the ligand specificity of each selectin. L-selectin-mediated rolling was drastically impaired if the cells lacked carbohydrate 6-sulfation elaborated by 6-Sul-T, but not affected when PSGL-1 was replaced with a mutant lacking three tyrosine residues at its NH(2) terminus. L-selectin-mediated adhesion was also hardly affected by mocarhagin treatment of the cells, which cleaved a short peptide containing sulfated tyrosine residues from PSGL-1. In contrast, P-selectin-mediated rolling was abolished when PSGL-1 was either mutated or cleaved by mocarhagin at its NH(2) terminus, whereas the cells expressing PSGL-1 and Fuc-T VII but not 6-Sul-T showed only a modest decrease in P-selectin-mediated adhesion. These results indicate that L-selectin prefers carbohydrate 6-sulfation much more than tyrosine sulfation, whereas P-selectin favors tyrosine sulfation in the PSGL-1 molecule far more than carbohydrate 6-sulfation. E-selectin-mediated adhesion was sulfation-independent requiring only Fuc-T VII, and thus the three members of the selectin family have distinct requirements for ligand sulfation.