Interleukin 1 (IL-1)-dependent melanoma hepatic metastasis in vivo; increased endothelial adherence by IL-1-induced mannose receptors and growth factor production in vitro.

BACKGROUND: The growth of cancer cells in inflammatory tissue is often observed. This can be the result of favorable conditions for endothelial cell adherence and/or increased production of local growth factors. PURPOSE: The role of the proinflammatory cytokine interleukin 1 (IL-1) in the prometastatic and growth-promoting environment of inflammation was studied in vivo, and the mechanism of cytokine action was studied in vitro as well. METHODS: Systemic inflammation was induced by the intravenous injection of IL-1 beta or lipopolysaccharide (LPS), and the hepatic metastasizing ability of B16 melanoma (B16) cells following intrasplenic injection was studied. IL-1 receptor blockade was accomplished with the use of the IL-1 receptor antagonist (IL-1Ra). In vitro, IL-1Ra was used to assess the mechanism for prometastasis and growth promotion of cultured hepatic sinusoidal endothelium stimulated with LPS. RESULTS: There was a statistically significant (P < .01) enhancement in the parameters of hepatic metastasis when B16 cells were injected intrasplenically either 4 hours after IL-1 injection or 6 or 12 hours after LPS injection. IL-1Ra pretreatment reduced IL-1-induced enhancement of metastasis by 73%-87% and completely inhibited the augmentation of metastasis following LPS injection. In vitro, the adherence of melanoma cells to LPS-treated endothelium increased nearly twofold but was completely abrogated when IL-1Ra was added before LPS. Similar to melanoma adherence, a 2.5-fold increase (P < .05) in functional mannose receptors was observed with LPS treatment but was prevented by the addition of IL-1Ra did not affect basal mannose-receptor activity in unstimulated epithelium. Mannose-receptor activity and B16 cell adherence significantly correlated (r = .9) with LPS treatment. Conditioned medium from LPS-stimulated epithelium augmented B16 cell proliferation compared with control conditioned medium (P < .01). Production of B16 cell growth factor(s) was markedly reduced (P < .01) when IL-1Ra was added. CONCLUSIONS: These results demonstrate that systemic inflammation induces an enhancement of melanoma cell metastasis and growth by IL-1-dependent mechanisms in vivo. In vitro, the mechanism(s) is consistent with IL-1-mediated increase in expression of mannose receptors and production of tumor cell growth factor(s) from the endothelium. IMPLICATIONS: Given the multiple and complex cytokine cascade induced in vivo and in vitro during LPS-induced systemic inflammation, IL-1 plays a strategic role. Since IL-1Ra is without side effects in humans, studies on intraoperative infusion of IL-1Ra during tumor resection may be indicated.

Interleukin 1-dependent and -independent mouse melanoma metastases.

BACKGROUND: The adhesion of cancer cells to the endothelial lining of blood vessels, which is important for metastasis, is promoted by the action of interleukin 1 (IL-1) and other cytokines. PURPOSE: IL-1-producing melanoma cells were used to induce metastases in mice to test whether melanoma metastasis--wherever it occurs--depends on the action of IL-1. METHODS: We used the following experimental designs in this study: 1) Male C57BL/6J mice were inoculated in the left cardiac ventricle with 5 x 10(4) murine B16 melanoma cells, and no treatment was given (control animals). 2) Mice received an intraperitoneal injection of either saline (control animals) or recombinant human IL-1 receptor antagonist (rHuIL-1Ra) 2 hours before the injection of cancer cells; thereafter, they received an additional injection of saline or rHuIL-1Ra daily for 20 days. 3) Mice received an intravenous injection of either saline or rHuIL-1Ra; 15 minutes later, mice that received saline were given either a second injection of saline (control animals) or an injection of bacterial lipopolysaccharide (LPS) to stimulate host IL-1 production and endothelial cell activation. The mice that received rHuIL-1Ra were also given an injection of LPS at this time. Six hours later, all mice were inoculated with cancer cells, followed by no further treatment. In all experiments, the mice were killed 20 days after the injection of cancer cells, and metastases were counted in multiple organs and bones. Metastasis incidence values (relating to the frequency that a given site was positive for metastasis) and metastasis development index values (relating to the extent of metastasis at a given site) were calculated. A hierarchical cluster analysis was performed to determine whether groups of organs exhibited characteristic changes in their metastasis development index values in response to the three treatments given (i.e., rHuIL-1Ra, LPS, or rHuIL-1Ra plus LPS). Reported P values are two-sided. RESULTS AND CONCLUSIONS: Treatment with rHuIL-1Ra alone significantly (P<.05) reduced the occurrence of metastasis in the bone marrow, spleen, liver, lung, pancreas, skeletal muscle, adrenal gland, and heart, indicating that host- and/or melanoma-derived IL-1 promoted metastasis in these organs; treatment with rHuIL-1Ra had no effect on metastasis in the kidney, testis, brain, skin, and gastrointestinal tract, suggesting that metastasis in these latter organs was IL-1 independent. Treatment with LPS alone significantly (P<.05) enhanced metastasis in the same organs for which rHuIL-1Ra treatment reduced metastasis, except for the heart and the adrenal gland. Treatment with rHuIL-1Ra 15 minutes before LPS treatment abrogated the LPS-mediated enhancement of metastasis. Two independent organ groups for which IL-1 promoted melanoma metastasis were identified in the cluster analysis.

Selective implantation and growth in rats and mice of experimental liver metastasis in acinar zone one.

The functional zonation of liver tissue provides a framework for studying the implantation and growth of metastatic colonies within given zones of the hepatic acini. A very exact method for calibrating the position of metastatic foci in the hepatic acini was made possible by using the succinate-dehydrogenase reaction, which reveals the functional differences of the acinar zones. In mouse livers, metastasis was induced by intrasplenic injection of both high and low metastatic-capacity B16 melanoma and Lewis lung carcinoma cells. In rats, liver metastasis resulting from rhabdomyosarcoma was studied by injecting these cells into the s.c. tissue. All cases of metastasis occurred in hepatic acinar zone 1, and no significant differences were detected resulting from the type of tumor, its metastatic potential, or the procedure used for obtaining the metastasis. In subsequent experiments, metastasis was induced after first altering the zonal distribution of the hepatic extracellular matrix; distribution of the sinusoidal macrophages; and the sinusoidal diameter. However, even under these conditions, metastasis continues to occur exclusively in hepatic acinar zone 1. Thus, metastatic predilection for hepatic acinar zone 1 cannot be explained solely in terms of hemodynamic causes or the influence of extracellular matrix. Although it may still turn out that these elements play some secondary role in the phenomenon, our research points to the sinusoidal endothelial cells as a factor directly responsible for metastatic predilection for zone 1.

Interleukin-1 receptor blockade reduces the number and size of murine B16 melanoma hepatic metastases.

Hepatic metastasis in melanoma is affected by processes of tumor cell adhesion to sinusoidal cells, avoidance of cytotoxic cells, and local growth-promoting activity. A role for interleukin-1 (IL-1) in these events was evaluated in vitro and in vivo by specifically blocking IL-1 receptors using the naturally occurring IL-1 receptor antagonist (IL-1Ra). At 10- and 100-fold molar excess, IL-1Ra reduced the IL-1-mediated adhesion of B16 melanoma cells to cultured hepatic sinusoidal endothelial cells by 60 and 100%, respectively. Conditioned media derived from murine hepatic sinusoidal endothelial cells contain growth-promoting activity for B16 cells, and IL-1 increased its production 2.5-fold (P < 0.01). The addition of IL-1Ra to sinusoidal cells reduced the spontaneous release of the growth-promoting activity by 32% (P < 0.05). In addition, blocking IL-1 receptors on melanoma cells reduced their responsiveness to endothelial-conditioned medium (P < 0.05). A single dose of IL-1Ra (0.5 mg/kg) given to mice i.p. 2 h prior to intrasplenic injection of melanoma cells reduced the number of hepatic metastases by 50% and the metastatic volume by 70% compared to vehicle-injected controls (P < 0.01). When given 2, 4, and 6 days after the injection of tumor cells, IL-1Ra reduced the volume of metastases by 58% (P < 0.01). Fifty % of mice pretreated with 0.5 mg/kg IL-1Ra and given 3 additional doses on days 2, 4, and 6 died after 13.5 +/- 0.4 days compared to 11.3 +/- 0.2 days for controls (P < 0.01). In mice pretreated and given 10 daily doses at 5 mg/kg, there was an 80% reduction in hepatic metastases. Using this regimen, survival was 18.1 +/- 2.4 days in the IL-1Ra group and 11.2 +/- 1.5 in controls (P < 0.001). These studies demonstrate a significant role for IL-1 in implantation and growth of metastatic melanoma in the liver.

Influence of host defenses on the hepatic colonization of B16F10 melanoma cells.

To investigate the significance of host immunity in metastasis we have simultaneously evaluated metastatic development and the tumoricidal action of host defenses in an experimental system for liver metastasis which involves the intrasplenic injection of B16F10 melanoma cells in syngeneic mice. In addition, three experimental groups were treated with immunosuppressive doses of cyclosporin A (CsA) during the following periods of the malignant process: 1st-5th days, 1st-12th days and 7th-12th days. Analysis of cytolytic effects of macrophages, NK cells and T-lymphocytes on tumor cells reveals a decay in antitumor immunity from the 7th day to the 12th day and a marked resistance of B16F10 melanoma cells derived from hepatic metastases to T-lymphocytes and NK cells. The 1st-5th day CsA treatment of tumor-bearing mice produced a reduction in both T-lymphocyte and macrophage reactions against tumor cells and a significant increase in the 7th day micrometastasis incidence in the liver. Once micrometastases have been established the CsA-treatment suppression on the 5th day allows the tumor growth rate in these mice to become the same as in controls. However, the 7th-12th day CsA treatment produces a clear inhibitory effect on focal metastatic development which may correspond to the in vitro antiproliferative effect of CsA, detected on cultured B16F10 melanoma cells.

Growth-associated changes in glutathione content correlate with liver metastatic activity of B16 melanoma cells.

B16 melanoma (B16M) was used to study the relationship between glutathione (GSH) metabolism and the metastatic activity of malignant cells. GSH content increased in B16M cells during the initial period of exponential growth in vitro, to reach a maximum of 37 +/- 3 nmol/10(6) cells 12 h after plating, and then gradually decreased to control values (10 +/- 2 nmol/10(6) cells) when cultures approached confluency. On the contrary, glutathione disulphide (GSSG) levels (0.5 +/- 0.2 nmol/10(6) cells) and the rate of glutathione efflux (GSH + GSSG) (2.5 +/- 0.4 nmol/10(6) cells per h) remained constant as B16M grew. Changes in enzyme activities involved in GSH synthesis or the glutathione redox cycle did not explain shifts in the glutathione status (GSH/GSSG). However, two facts contributed to explain why GSH levels changed within B16M cells: a) high intracellular levels of GSH induced a feed-back inhibition of its own synthesis in B16M cells from cultures with low cellular density (LD cells); b) transport of cyst(e)ine, whose availability is the major rate-limiting step for GSH synthesis, was limited by cell-cell contact in cultures with high cellular density (HD cells). Intrasplenic injection of B16M cells with high GSH content (exponentially-growing cultures) showed higher metastatic activity in the liver than cells with low GSH content (cells at confluency). However, when low GSH-content cells (HD cells) were incubated in the presence of GSH ester, which rapidly enters the cell and delivers free GSH, their metastatic activity significantly increased. Our results demonstrate that changes in GSH content regulate the metastatic behaviour of B16M cells.

Theophylline administration markedly reduces hepatic and pulmonary implantation of B16-F10 melanoma cells in mice.

Theophylline-treated B16-F10 melanoma cells show a lower experimental metastatic potential in vivo. To identify the possible mechanism(s) involved and on the basis of previous reports, we tested the induction of apoptosis in B16-F10 cells. Fluorescence activated cell sorter (FACS) analysis and p53 overexpression in theophylline-treated B16-F10 melanoma cells appeared to suggest enhanced cell death by apoptosis. The in vivo effects of orally administered theophylline in mice were investigated using different treatment schedules in mice that had undergone hepatic or pulmonary colonization with tumour cells. Mice received theophylline in their drinking water according to different protocols: (i) from 3 days before tumour cell inoculation until animal sacrifice ('early treatment'); (ii) from 3 days before until 3 days after tumour cell inoculation ('short treatment'); or (iii) from 3 days after tumour cell inoculation until animal sacrifice ('late treatment'). In the 'early treatment' group, the number of melanoma foci was reduced by 92.3% in the liver and 81.4% in the lung compared with control animals (P < 0.001). In the 'short treatment' group, there was an 80.2% and 72.2% reduction in liver and lung metastases, respectively (P < 0.001). In the 'late treatment' group, the inhibition of metastasis was 59.7% for liver and 45.3% for lung (P < 0.005). Survival studies showed that 50% of the 'early' theophylline-treated animals died 33.2 +/- 2.0 days after intrasplenic injection (control group: 23.1 1.8 days; P < 0.001) and 33.9 +/- 2.5 days after tail vein injection (control group: 24.1 +/- 1.4 days; P < 0.001). Taken together, these observations provide useful information for the potential clinical application of theophylline as a chemotherapeutic agent against malignant melanoma.

Sinusoidal structure of the liver.

Following a review of current information on the structural and functional characteristics of liver tissue with view to explaining the heterogeneity of the structure of the sinusoids within each microcirculatory unit, we analyze the zone-specific properties of hepatic parenchymal and non-parenchymal cells. However, in addition to zonal differentiation of hepatocytes with respect to their metabolic activities, we also discuss the appreciable zonal variations along the sinusoidal pathway in the caliber and hemodynamics of sinusoids, in its fenestration pattern, endocytic and macrophagic efficiency and interactions with circulating normal and tumor cells. This leads us to the conclusion that sinusoidal cells diversify functionally in each domain of the hepatic acinus, showing distinct specific properties in each segment of the sinusoidal path.

Fenestration patterns in endothelial cells of rat liver sinusoids.

Endothelial fenestrae of both zone 1 and zone 3 acinar liver sinusoids have been studied in rats by an interactive analysis of scanning electron microscopical images. Two fenestration patterns have been recognized in the endothelial cells on the basis of local variation in size, distribution and clustering of pores in each acinar zone. Our data indicate that both the number of fenestrae per square micrometer of endothelial surface and the mean diameter of fenestrae are significantly larger in zone 3 than in zone 1. The number of sieve plates is about 1.74 times larger in zone 3 than in zone 1, and the number of fenestrae per plate in zone 3 is nearly twice that in zone 1. Two different classes of fenestrae have been considered: clustered pores, which prevail in zone 3 and have a mean diameter smaller than the other pores, and free pores, which prevail in zone 1 and are bigger.

Enalaprilat inhibits hydrogen peroxide production by murine mesangial cells exposed to high glucose concentrations.

BACKGROUND: Oxidative stress is considered to play a role in the pathogenesis of diabetic nephropathy. Angiotensin-converting enzyme (ACE) inhibitors are hypotensive drugs with a well-known effect in preventing the progression of chronic renal failure. Their mechanism of action is not clearly established. METHODS: The effect of enalaprilat on hydrogen peroxide (H2O2) production by cultured murine mesangial cells exposed to 5.5 (basal condition), 30 and 50 mM glucose concentrations was examined over 8 h. A fluorimetric method quantifying, in arbitrary units, the intracellular dichlorofluorescein (DCFH) oxidation to the highly fluorescent compound 2'7'dichlorofluorescein (DCF) from the non-fluorescent probe dichlorofluorescein-diacetate (DCFH-DA) was employed (a method not previously reported for cultured mesangial cells). Experiments were repeated three times in quadruplicate wells. RESULTS: H2O2 production by mesangial cells exposed to 50 mM glucose was significantly increased after 1 h, compared to cells exposed to 5.5 and 30 mM glucose. This observation was not reproduced with 50 mM mannitol. Addition of 100 ng/ml enalaprilat to cells with 50 mM glucose significantly inhibited H2O2 production during the 8 h of the assay. This response was similar to that obtained with 100 ng/ml catalase. Increasing enalaprilat concentrations (10, 50 and 100 ng/ml) also significantly decreased the constitutive H2O2 generation in the presence of 5.5 mM glucose. Angiotensin II and saralasin, both at 1 microM, did not modify H2O2 production by cells exposed to 5.5 mM glucose. In contrast, 1 microM staurosporine, a protein kinase C (PKC) antagonist, significantly decreased H2O2 generation in the presence of 50 mM glucose. CONCLUSION: Enalaprilat has an antioxidant effect in cultured mesangial cells. This action is not linked to ACE inhibition, but may be related to an inhibition of the PKC system.

Differential location of hemopoietic colonies within liver acini of postnatal and phenylhydrazine-treated adult mice.

We have measured the location of embryonic and adult hemopoietic foci in the liver tissue of postnatal and adult phenylhydrazine-treated mice. Differentiation of acinar domains in liver tissue was made possible by carrying out succinate dehydrogenase histochemical reactions on liver cryostat sections. To determine the position of hemopoietic foci within the lobular gradient of the hepatocyte succinate dehydrogenase activity, this enzyme was measured in hepatocytes surrounding both portal and central veins and hemopoietic foci. Then, assuming the periportal succinate dehydrogenase activity value to be 1.00 +/- 0.2, succinate dehydrogenase activity around postnatal hemopoietic foci was 0.65 +/- 0.19, around phenylhydrazine-induced hemopoietic foci 0.83 +/- 0.24 and around central veins 0.44 +/- 0.11. Scaling the portal to central vein distance and taking 1 as the portal vein point and 0 as the central vein point, the relative position of hemopoietic foci, indirectly calculated from succinate dehydrogenase activity values, was 0.35 +/- 0.13 in postnatal livers and 0.73 +/- 0.12 in phenylhydrazine-treated adult livers. Hemopoietic foci frequencies varied according to both the origin and the liver acinar domain: in postnatal liver acini, it was 37.1% in zone 1, 22.8% in zone 2 and 40% in zone 3; in phenylhydrazine-treated adult acini, it was 89.4% in zone 1 and 10.6% in zone 2. Postnatal hemopoietic foci mainly occurred extrasinusoidally, between hepatocytes and reticular-like cells, whereas adult hemopoietic foci were mostly intrasinusoidal and closely associated to macrophage-like cells. Adult hemopoietic colonies

Interleukin-1-mediated H2O2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion.

We have examined H2O2 production by in vitro enriched hepatic sinusoidal endothelium (HSE) during interleukin-1 beta (IL-1 beta) stimulation and B16 melanoma cell adhesion. Production of H2O2 was quantified by flow cytometry and multiwell plate-scanning fluorimetry of intracellular 2', 7'-dichlorofluorescein (DCFH) oxidation in HSE. Under IL-1 beta treatment there was a 6-fold increase in endothelial cells producing H2O2 (67%) and a 4-fold augmentation in the Kupffer cell population (86%). The average H2O2 content per cell size unit significantly (P < 0.01) increased in endothelial cells (2.6-fold) and Kupffer cells (1.7-fold). In contrast to the homogeneity of Kupffer cells, H2O2 production intensity was largely heterogeneous in IL-1 beta-activated HSE. Enhancement of H2O2 production by IL-beta-treated HSE started at the 4th h and peaked 2-3 h later. The addition of increasing concentration of IL-1 beta to HSE for 4 h caused the progressive activation of H2O2 production by treated cells. The addition of 80 M excess of IL-1 receptor antagonist (IL-1 Ra) 10 min before IL-1 beta treatment abrogated IL-1 beta-mediated enhancement of H2O2. From the 2nd h of B16 melanoma adhesion to HSE there was significantly (P < 0.05) enhancement of H2O2 content in HSE. This activation increased 2.25-fold by the 3rd h of coculture and had reduced again by the 5th h. IL-1 Ra (80 ng/ml) given to HSE 10 min before melanoma cells abrogated the HSE response to melanoma cells. The addition of 1% paraformaldehyde (PFA)-fixed B16 melanoma cells to HSE did not affect H2O2 production response, indicating that HSE-activating agents were on the melanoma cell surface. Preincubation of B16 melanoma cells in the presence of 5 micrograms/ml anti-mouse IL-1 beta neutralizing antibody reduced the melanoma cell-induced HSE production of H2O2 by 80%. On the contrary, B16 melanoma cell-conditioned medium did not vary HSE production of H2O2 compared to control HSE. Western blot analysis of cytosolic and membrane sediments from B16 melanoma cells confirmed the presence of IL-1 beta (17.4 kDa) in both cell compartments. Thus, HSE responded to melanoma cell contact with a rapid production of H2O2. HSE activation was IL-1-dependent. This cytokine was directly provided to HSE by the cell surface of adhered melanoma cells.

Hepatic sinusoidal endothelium heterogeneity with respect to mannose receptor activity is interleukin-1 dependent.

Using fluorescein isothiocyanate-conjugated ovalbumin (OVA-FITC), 125I-mannan, or 125I-invertase as specific ligands for the mannose receptor, we have quantified its activity in mouse and rat hepatic sinusoidal endothelium (HSE), under both basal conditions and after lipopolysaccharide (LPS) or human recombinant interleukin-1beta (IL-1beta) stimulations. Mouse treatment for 4 hours with 5 microg/kg IL-1beta significantly increased OVA-FITC uptake by HSE. Ligand uptake exhibited a sublobular compartmentalization: In control mice as well as in IL-1beta-stimulated mice, the ligand distributed preferentially in the periportal and septal areas; no OVA-FITC was observed in the perivenous sinusoids. In vitro exposure of mouse HSE to 100 pg/mL LPS or 1 ng/mL IL-1beta for 6 hours significantly (P < .01) increased OVA-FITC uptake. Blocking IL-1 receptors in HSE by addition of 100 ng/mL IL-1 receptor antagonist (IL-1Ra) before stimulation with LPS or IL-1beta abrogated the increase in mannose receptor-mediated uptake. In vitro endocytosis assays showed that rat HSE uptake of 125I-mannan or 125I-invertase progressively increased with both exposure time and concentration of added IL-1beta. Upregulation of mannose receptor-mediated uptake in response to IL-1beta or LPS was also blocked by previous addition of IL-1Ra to rat HSE. Flow cytometric analysis showed a significant HSE heterogeneity in mannose receptor-mediated endocytosis in response to IL-1beta treatment: type I endothelial cells (EC-I, defined by their small size and high cytoplasmic density) significantly (P < .01) increased OVA-FITC uptake compared with type II endothelial cells (EC-II, defined by their large size and low cytoplasmic density). In addition, the subset of EC-I contained three times more IL-1beta-binding cells than the EC-II subset. Because EC-I and EC-II are preferentially located in the periportal and perivenous segments of hepatic sinusoids, respectively, these results suggest that IL-1beta, apart from upregulating mannose receptor activity, contributes to the sublobular compartmentalization of this endothelial cell function.

Quantitative microscopy of mouse colon 26 cells growing in different metastatic sites.

Quantitative microdensitometry and computerised interactive image analysis were used to compare the expression of endogenous lectins by cells of mouse colon 26 carcinomas, growing either as primary tumours or metastases, in five different anatomic sites (caecum, liver, lung, spleen, s.c.). Endogenous lectins were visualised in tissue sections using the ABC peroxidase technique with a panel of 17 biotinylated neoglycoproteins representing a variety of carbohydrates found in glycoproteins, glycolipids and proteoglycans. Clear-cut site-associated differences in endogenous lectin expression were detected in cancer cells growing in all five sites. The patterns of these changes were complex and shifts in expression of different lectins were independently variable in both direction and amount. In addition to site-associated variations, differences in lectin expression were also detected in the liver and lungs, between cells in spontaneous metastases and cells in colonies generated by direct injection of cancer cells into the bloodstream. The results demonstrate quantitative, as distinct from qualitative, differences developing in cancer cell populations after delivery of cells to different target organs. The differences between liver and lung metastases are in accord with analogous site-associated differences in metastatic patterns produced by colon carcinoma cells in mice and in humans.

Differences in the lectin-binding patterns of the periportal and perivenous endothelial domains in the liver sinusoids.

We have studied the distribution patterns of carbohydrate terminals on the endothelial surface of the mouse liver microvasculature. For this purpose, a wide battery of FITC lectins specific to glucose, mannose, galactose, fucose, N-acetyl-neuraminic acid, N-acetyl-galactosamine and N-acetyl-glucosamine residues were incubated on liver cryostat sections or intraportally perfused under physiological conditions. All the resulting hepatic sections were examined under fluorescent microscopy and confocal laser scanning microscopy. With the exception of N-acetyl-galactosamine- and fucose-binding lectins, all the perfused lectins specifically bound to the microvascular wall as confirmed by blocking methods using their corresponding sugars. A wide range of binding was, however, observed among the lectins, and the latter were classified into four groups according to their affinities for the different segments of the hepatic microvasculature: (a) equal affinity for all segments (concanavalin A); (b) different affinities depending on acinar zone (wheat germ agglutinin, Ricinus communis toxin, phytohemagglutinin E, Erythrina cristagalli agglutinin and Pisum sativum agglutinin); (c) preferential binding to the sinusoidal network (Lathyrus odoratus, phytohemagglutinin); and (d) lectins that fail to bind to the hepatic microvasculature (N-acetyl-galactosamine- and fucose-binding lectins). Sinusoidal segment walls in acinar zone 1 expressed a higher concentration of certain lectin-binding carbohydrate residues (N-acetyl-neuraminic acid, N-acetyl-galactosamine, galactose, mannose and glucose) than in acinar zone 3. The labeling patterns obtained through the incubation of liver sections or through in vivo perfusion with the different lectins did not always coincide. Only concanavalin A, wheat germ agglutinin and phytohemagglutinin E lectins proved to be concordant (i.e., they produced identical labeling patterns in both procedures).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and enrichment of two sublobular compartment-specific endothelial cell subpopulations from liver sinusoids.

Similar to the well-recognized phenotypical heterogeneity of hepatocytes, in situ sublobular variations have recently been detected in the cell structure, fenestration patterns, filtrating efficiency, surface glycosylation, scavenger function and pathological responses of the sinusoidal lining endothelium. However, unlike other liver cell populations, until now no endothelial cell subpopulations had been isolated or defined with clarity, much less with sublobular/acinar zone-related differential properties. On the basis of our previous studies showing that periportal segments of mouse liver sinusoids express a significantly higher number of wheat germ agglutinin-binding sites than do perivenous ones, we used this differential feature for in vitro labeling of the specific sublobular derivation of isolated sinusoidal lining endothelial cells to correlate their original lobular position with other features determined on flow cytometry, centrifugal elutriation, discontinuous arabinogalactan density gradients and electron microscopy. Our results revealed additional heterogeneous properties whose association with high or low wheat germ agglutinin-binding capacity made it possible to define in vitro two dominant endothelial cell subpopulations that appear similar to the differential features in the periportal and perivenous sinusoidal segments. Type 1 endothelial cells had low forward angle light scatter and high integrated side scatter, low cytoplasmic porosity index (12% +/- 5%) and high wheat germ agglutinin-binding efficiency (160 +/- 35 fluorescence intensity units/cell size); these findings are similar to what was observed in situ in the periportal sinusoidal endothelium. On the other hand, type 2 endothelial cells, with high forward angle light scatter and low integrated side scatter, had a high cytoplasmic porosity index (25% +/- 8%) and low wheat germ agglutinin-binding efficiency (60 +/- 15 fluorescence intensity units/cell size), findings similar to in situ observations of the perivenous sinusoidal lining endothelium. Moreover, these physical and morphological differences entail different cell sedimentation behaviors: type 1 endothelial cell sedimented at high centrifugal elutriation counterflow rates (23 to 37 ml/min) and high arabinogalactan density gradient levels (10% to 15%), whereas type 2 endothelial cell sedimented at low counterflow rates (18 to 23 ml/min) and low density levels (6% to 10%). The combination of these separation procedures made it possible to isolate a 90%-enriched type 1 endothelial cell population in the 12% to 15% interphase of the 23 and 37 ml/min elutriation flow rates and a 75%-enriched type 2 endothelial cell population in the 6% to 10% interphase of the 18 and 23 ml/min flow rates.

Differential cytokine-mediated modulation of endocytosis in rat liver endothelial cells.

We have studied the effects of exogenous tumor necrosis factor-alpha, interleukin-1 beta, and lipopolysaccharide-induced interleukin-1 beta on receptor-mediated endocytosis in primary cultures of liver sinusoidal endothelial cells. Tumor necrosis factor-alpha and interleukin-1 beta enhanced endocytosis via the scavenger and mannose receptors in a dose-time dependent manner, while the function of the collagen receptor remained unaffected. The modulatory effects of the cytokines were blocked by adding specific inhibitors for both cytokines. Results from studies on binding (4 degrees C) and internalization and degradation (37 degrees C) of ligands indicate that the increased scavenger capacity of liver sinusoidal endothelial cells resulting from exposure to the inflammatory mediators was due to increased rate of intracellular transport of endocytosed ligands to lysosomes, rather than to increased binding to receptors.

Tumor-dependent activation of rodent hepatic stellate cells during experimental melanoma metastasis.

In this work we report the presence of intrametastatic smooth-muscle iso-alpha-actin (SMA)-expressing cells which appeared from the early stages of the hepatic metastasis process of intrasplenically injected B16 melanoma (B16M) cells. They formed a network of stromal cells among B16M cells, a very low percentage of them expressing desmin. In contrast, those parts of liver tissue unaffected by metastasis had perisinusoidal desmin-expressing quiescent hepatic stellate cells (qHSC) which did not express SMA. Exposure of freshly isolated rat quiescent hepatic stellate cells (qHSC) to B16M cell-conditioned medium (B16M-CM) leads to a progressive increase (P < .01) in the number of SMA-expressing cells, which was accompanied by a parallel reduction in the number of desmin-expressing cells. In addition, B16M-CM also contained chemotactic factor(s) which significantly (P < .01) increased (50%) in vitro qHSC migration and stimulated both [3H]thymidine and [3H]glucosamine uptake in qHSC. Moreover, B16M-CM also significantly (P < .01) enhanced qHSC secretion of matrix metalloproteinase-2 (MMP-2), and unknown chemotactic factor(s) enhancing in vitro migration of B16M cells. The results suggest that B16 melanoma releases qHSC-activating factors, which induce the appearance of metastasis-infiltrating myofibroblasts by a paracrine mechanism. Such cells showed cytoskeletal alterations which are associated with enhanced proliferation, glycosaminoglycan synthesis, MMP-2 secretion, and tumor-chemotactic factor production. Thus, tumor-activated qHSC may play an important role in melanoma cell motility and invasion during hepatic metastasis progression.

Mannose receptor-mediated endothelial cell activation contributes to B16 melanoma cell adhesion and metastasis in liver.

The role of mannose receptors from hepatic sinusoidal endothelium (HSE) in liver colonization by B16 melanoma (B16M) cells was studied. The expression of high mannose-type oligosaccharides on the surface of B16M cells was enhanced by in vitro treatment with 1-deoximannojirimycin (1-DMM). There was a significant (P < 0.01) enhancement of hepatic metastasis when B16M cells were 1-DMM-treated before being intrasplenically injected into C57BL/6J mice. Intraperitoneal administration of 5 mg/kg recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) inhibited the 1-DMM-induced enhancement of metastasis. Expression of high mannose-type oligosaccharides on the surface of 1-DMM-treated B16M cells and their in vitro adhesion to the HSE was significantly correlated (R = 0.82). The addition of either 100 microg/ml mannan or paraformaldehyde (PFA)-fixed 1-DMM-treated B16M cells to cultured HSE for a period of 12 h significantly (P < 0.01) increased the release of IL-1beta from the HSE compared to that liberated by the HSE incubated with either basal medium or PFA-fixed untreated B16M cells. The same HSE treatments also significantly (P < 0.01) increased the degree of adhesion of other B16M cells to HSE, being abrogated by anti-mouse vascular cell adhesion molecule-1 (VCAM-1) antibodies. The conditioned media from HSE cultures, activated by PFA-fixed, 1-DMM-treated B16M cells significantly (P < 0.01) increased B16M cell proliferation when compared to conditioned media from HSE cultures incubated with PFA-fixed, untreated B16M cells. Thus, 1-DMM treatment of B16M cells enhanced the development of hepatic metastasis by IL-1-dependent mechanisms. The mechanism is consistent with in vitro mannose receptor-mediated melanoma cell attachment to the HSE, which subsequently upregulates IL-1beta release, VCAM-1-dependent adherence, and melanoma growth factor(s) release by HSE.

Coincident implantation, growth and interaction sites within the liver of cancer and reactive hematopoietic cells.

We have examined the anatomical-functional sites within mouse liver where phenylhydrazine (PHZ)-induced hematopoietic foci, and M5076 reticulum cell sarcoma, B16F10 melanoma and Lewis lung-carcinoma cells specifically develop as colonies after intrasplenic injection. Cancer foci occurred predominantly in the 2.4 to 4.0 segment of the sinusoidal pathway, corresponding to hepatic acinar zone I. No significant differences were detected between different types of tumor, including their different tendencies to spontaneously metastasize liver, or as a result of the different procedures used for obtaining foci or metastases. In addition, PHZ-treatment of mice previously injected with tumor cells, resulted in double colonization of the liver tissue by both hematopoietic and cancer cells, predominantly in zone I. This spatial coincidence indicates that non-cancer-specific mechanisms operate in zone I, either promoting implantation and/or growth of cell colonies or, alternatively, inhibiting these processes in the region surrounding the central vein (Rappaport zone 3). Our observations failed to reveal mutual displacement of cancer or hematopoietic foci by potential competition for development sites in zone I. Enumeration and diameter measurements of cancer foci in PHZ-treated animals showed that the presence of hepatic hematopoietic foci coincided with a significant increase in the hepatic metastasis volume. However, the fact that no significant differences in pulmonary metastases occurred in both the PHZ-treated and control mice given tail-vein injection of cancer cells, and that PHZ reduces cancer cell proliferation in vitro, reveal evidence of local interactions with hematopoietic foci which promote growth of cancer foci in liver.