Malignant pleural mesothelioma presenting as achalasia.

A 65-year-old man with an occupational history of asbestos exposure developed dysphagia and vomiting. Clinical examinations at onset revealed a dilated esophagus with smooth narrowing at the gastroesophageal junction and no apparent tumor in and around the esophagus. Achalasia was suspected. Dysphagia progressed gradually and examinations performed three months after the onset disclosed a tumor in the pleural and the peritoneal cavities. At laparotomy, the tumor extended from the pleural cavity into the peritoneal cavity. Histological examination of the biopsied specimen demonstrated malignant mesothelioma. We report the first case of malignant pleural mesothelioma presenting as achalasia.

Kupffer cells inhibit the lymphoproliferative response to antigenic stimulation by rat hepatocytes.

Sprague-Dawley rats were immunized with liver-specific protein. Kupffer cells and hepatocytes were prepared by the enzyme digestive procedure, and macrophage-depleted lymphocytes were prepared by the glass adherence method. Lymphocytes were incubated with macrophages or with Kupffer cells during the antigenic stimulation of mitomycin-C-treated hepatocytes for 90 h, and 3H-thymidine incorporation of lymphocytes was analyzed. The proliferation of lymphocytes cultured with macrophages was increased, but the proliferation of lymphocytes cultured with Kupffer cells was decreased in the rats immunized with liver-specific protein. And cytotoxicity to hepatocytes was increased after lymphocyte-Kupffer cell culture in the immunized rats. On the other hand, the proliferation of lymphocytes and cytotoxicity to hepatocytes were not significantly increased in normal rats. In the immunized rats lymphocytes cultured with macrophages showed a proliferative response to the membrane of hepatocytes, but inhibition of the lymphoproliferative response by Kupffer cells is considered to be due to destruction of hepatocytes (and thereby removal of antigen).

Kupffer cell function in chronic ethanol-fed rats.

In an attempt to clarify the Kupffer cell function in alcoholism, chronic ethanol-fed rats were investigated. The clearance of latex particles in the rat was analysed to estimate the function of the reticuloendothelial system in the liver, and the phagocytic function of Kupffer cells was measured by counting particles in the cell after isolation of non-parenchymal cells by collagenase digestion of the liver following an injection of latex particles and subsequently by staining of endogenous peroxidase activities. In addition, the number of Kupffer cells and their phagocytic function were examined histologically in fresh frozen sections of liver after an injection of particles. Serum ethanol concentration in the ethanol-fed rats was 10-60 mumol/l. The clearance of latex particles was markedly reduced in the ethanol-fed rats as compared with the paired controls (P less than 0.01). Markedly decreased-phagocytic function was found in 20% of Kupffer cells in the chronic ethanol-fed rats. The number of Kupffer cells in the ethanol-fed rats was increased as compared with the paired control rats. Chemotaxis analysis revealed that hepatocytes when incubated with ethanol, produced chemotactic factor for Kupffer cells and polymorphonuclear cells. These abnormal Kupffer cell functions may contribute to the pathogenesis of alcoholic liver disease.

Modulation of ischemia-reperfusion-induced hepatic injury by Kupffer cells.

To elucidate the role of Kupffer cells in ischemia-reperfusion-induced hepatic injury, hepatic injury induced by ischemia-reperfusion was analyzed after modulation of Kupffer cell function. Ischemia of the liver was performed by occlusion of both the portal vein and hepatic artery, which enter into the left lateral and median lobes of the liver. Blood flow in the ischemic lobe was reduced, in contrast to an increased blood flow in the nonischemic lobe during occlusion of the veins. Although hepatocyte damage was not demonstrated by ischemia for < 60 min, hepatic injury was found after reperfusion of the liver, and activation of Kupffer cells was morphologically demonstrated by electron microscopies. Suppression of Kupffer cells, induced by previous administration of gadolinium chloride or latex particles, reduced the grade of hepatic injury induced by ischemia-reperfusion. On the other hand, stimulation of Kupffer cell phagocytosis, induced by administration of latex particles at the time of reperfusion, aggravated the ischemia-reperfusion-induced hepatotoxicity, which was then reduced by simultaneous administration of superoxide dismutase. Kupffer cells, isolated from the rats treated with the ischemia-reperfusion procedure, have been found to release increased amounts of oxygen radical intermediates. These results suggest that hepatic injury induced by ischemia-reperfusion is modulated by the function of Kupffer cells and that superoxide anion released from Kupffer cells could play an important role in ischemia-reperfusion hepatic injury.

Endotoxin induced cellular communication in the liver: murine models for clarification of the role of LPS-responsive macrophages in the pathogenesis of liver diseases.

In several experimental models, lipopolysaccharide (LPS) plays an important role in the pathogenesis of liver diseases. Murine models of C3H/HeN and C3H/HeJ mice have been used to elucidate the role of LPS and its responsive-macrophages in vivo, as C3H/HeN strain mice are known to be LPS-responsive, while C3H/HeJ strain mice are LPS-resistant. Furthermore, release of several kinds of biologically active mediators such as interleukin-1, tumour necrosis factor-alpha, colony stimulating factor and reactive oxygen radical is not enhanced in C3H/HeJ mice even after stimulation with LPS. Thus, these murine models could be suitable for clarification of endotoxin induced cellular communication in the liver.

Phagocytic properties of hepatic endothelial cells and splenic macrophages compensating for a decreased phagocytic function of Kupffer cells in the chronically ethanol-fed rats.

The balance of phagocytic function among Kupffer cells, hepatic endothelial cells and splenic macrophages in the chronically ethanol-fed rats has been investigated. Clearance of latex particles in the blood was measured to estimate the function of the reticuloendothelial system. Phagocytosis of latex particles by Kupffer cells, hepatic endothelial cells or splenic macrophages in vivo was measured by counting the number of ingested particles in a cell after isolation of hepatic nonparenchymal cells or spleen cells following injection of different amounts of latex particles. Latex particle clearance was suppressed in the ethanol-fed rats, demonstrating a decreased phagocytic capacity of the reticuloendothelial system. Markedly decreased phagocytic function was found in 40% of Kupffer cells of the chronically ethanol-fed rats. In contrast, the number of latex particles in hepatic endothelial cells and in splenic macrophages was increased after injection of a triggering dose of latex particles. From these results it may be concluded that an increased phagocytosis of hepatic endothelial cells and splenic macrophages could compensate for the decreased phagocytic function of Kupffer cells.

Generation of chemotactic factor by hepatocytes isolated from chronically ethanol-fed rats.

In an attempt to clarify a mechanism of polymorphonuclear cell and/or macrophage infiltration in alcoholic liver disease, we investigated a novel chemotactic and activating factor generated by rat hepatocytes isolated from the chronically ethanol-fed rats. Hepatocytes and hepatic macrophages were isolated from rat liver by perfusion and digestion with collagenase and subsequently by differential centrifugation on a metrizamide gradient. Rat polymorphonuclear cells were prepared from blood by the dextran sedimentation and Hypaque-Ficoll technique. Chemotactic activity was measured as migration of polymorphonuclear cells or hepatic macrophages using a chemotactic chamber. When hepatocytes isolated from the ethanol-fed rats were cultured in vitro, chemotactic activity for rat polymorphonuclear cells and hepatic macrophages was demonstrated in the culture supernatant. Inhibitors of transcription and protein synthesis reduced generation of chemotactic factor from these hepatocytes. Chemotactic activity of the conditioned medium was reduced after trypsin (0.25%, 37 degrees C, 30 min) or heat (56 degrees C, 30 min) treatment. The chemotactic activity was eluted at molecular weights of 20-25 kDa and 40-45 kDa following Sephadex G-150 chromatography. Superoxide anion production by polymorphonuclear cells and hepatic macrophages under the stimulation of phorbolmyristate acetate was enhanced in the presence of this chemotactic factor. This chemotactic factor may contribute to the pathogenesis of alcoholic liver disease.

Modulation of hepatotoxicity by macrophages in the liver.

In an attempt to elucidate the role of hepatic macrophages in liver injury, we investigated galactosamine-treated rats (500 mg per kg body weight). The rats received an i.v. injection of latex particles (2 x 10(9) particles per animal) prior to (latex-galactosamine) or 12 to 16 hr subsequent to the galactosamine treatment (galactosamine-latex). Effect of superoxide dismutase on hepatic injury induced by galactosamine or galactosamine-latex treatment was also examined. Oxygen-derived free radical-generating capacity of isolated hepatic macrophages was measured as chemiluminescence with the stimulation of phorbol myristate acetate or latex particles. As compared with normal rats, chemiluminescence of hepatic macrophages from galactosamine-treated rats was 5- to 10-fold enhanced 12 hr following galactosamine treatment and remained elevated for 48 hr. Chemiluminescence of the latex particle-pretreated macrophages in the liver was markedly suppressed even following the galactosamine treatment (p less than 0.01). Compared to galactosamine-treated rats, both lipid peroxide level in the liver tissue and AST and ALT concentration in serum were significantly decreased in the latex-galactosamine-treated rats (p less than 0.01) and increased in the galactosamine-latex-treated rats (p less than 0.01). Furthermore, superoxide dismutase supplementation protected against liver injury induced by the galactosamine-latex treatment. From these results, pretreatment with latex particles suppressed the free radical-generating capacity of hepatic macrophages and protected against hepatic injury induced by galactosamine. In contrast, injection of latex particles after galactosamine treatment aggravated hepatic injury, which was prevented by superoxide dismutase. These data suggest that liver injury induced by galactosamine is modulated by oxygen-derived free radicals from hepatic macrophages.

Mechanism of accumulation of macrophages in galactosamine-induced liver injury: effect of lipoxygenase inhibitors on chemotaxis of spleen cells.

In an attempt to clarify a mechanism of macrophage infiltration in galactosamine-induced hepatic injury, we investigated chemotactic factor(s) generated by murine hepatocytes exposed to galactosamine. Hepatocytes, isolated from murine liver by perfusion and digestion with collagenase, were incubated with galactosamine. Conditioned medium was collected 24 h later and chemotaxis of murine spleen cells was measured by stimulation of the conditioned medium using a modified Boyden chamber. Chemotactic activity was demonstrated in the conditioned medium of hepatocytes exposed to more than 3 mM galactosamine. Chemotactic activity of the conditioned medium was not reduced after freeze-thawing, and found to be dialyzable (molecular weight < 12,000). Trypsin (0.25%, 37 degrees C, 30 min) or heat (56 degrees C, 30 min) treatment reduced chemotactic activity of the conditioned medium. Furthermore, chemotaxis of spleen cells was decreased in the presence of lipoxygenase inhibitors (azelastine, ketotifen). These results suggest that accumulation of macrophages in the liver could be mediated by chemotactic factor produced by the galactosamine-treated hepatocytes, and that this mechanism may contribute to the pathogenesis of hepatic injury induced by galactosamine.

Hepatocyte nitric oxide production is induced by Kupffer cells.

To investigate the cellular communication in the liver, nitric oxide (NO) production by sinusoidal cells and hepatocytes by stimulation with cytokines and Kupffer cell-conditioned medium was quantitatively analyzed. NO production by the cells was measured by the Griess reaction, and nitric oxide synthase (iNOS) transcription level by a competitive RT-PCR assay using mutant iNOS mRNA as a standard. NO production and iNOS mRNA transcriptional levels in Kupffer cells were markedly increased by stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), and moderately by interleukin-1beta (IL-1beta). NO production by hepatocytes was not significantly enhanced by LPS, but was markedly enhanced by IL-1beta or the combination of tumor-necrosis factor-alpha (TNF-alpha) and IFN-gamma. Hepatocyte NO production and iNOS mRNA levels were markedly enhanced by the LPS-activated Kupffer cell conditioned medium, but these effects were reduced by heat treatment or anti-TNF antibody. Although NG-monomethyl-L-arginine acetate and dexamethasone reduced NO production by the cells, the iNOS mRNA level was reduced by dexamethasone only. Gel-shift assay showed NF-kappaB activation in hepatocytes during this activation. These data reinforce the importance of cellular communication between sinusoidal cells and hepatocytes.

Chemotactic factors released from hepatocytes exposed to acetaminophen.

To clarify the mechanism of neutrophil infiltration in the liver of acetaminophen-induced hepatic injury, chemotactic factor released from hepatocytes exposed to acetaminophen has been investigated. Hepatocytes exposed to acetaminophen release nondialyzable chemotactic factor, although acetaminophen in itself inhibits chemotaxis of neutrophils. Chemotactic activity of the nondialyzable chemotactic factor was reduced after treatment with heat (56 degrees C, 30 min) or trypsin. Chemotactic activity was demonstrated at the molecular weights of around 25 and 55 kDa. Chemotactic activity of the conditioned medium was not significantly reduced in the presence of antibody against rat KC/gro protein (interleukin-8-related cytokine in rodent). Chemotactic activity of a 25-kDa factor was reduced by the antibody against the antibody against KC/gro protein, but that of a 55-kDa factor was not reduced. Immunoblot analysis revealed that the peptide reacted with antibody against rat KC/gro protein was demonstrated at a molecular weight of around 20-25 kDa, but not around 55kDa, when the conditioned medium of acetaminophen-treated hepatocytes was electrophoresed. These results suggest that hepatocytes exposed to acetaminophen release two types of chemotactic factors for neutrophils and that a major part of the chemotactic factor could be different from a member of interleukin-8 family.

Oxygen-derived free radical generating capacity of polymorphonuclear cells in patients with ulcerative colitis.

Oxygen-derived free radical generating capacity of polymorphonuclear cells in 27 patients with ulcerative colitis, 10 with acute bacterial diarrhea and 20 healthy volunteers, was measured by the luminol-dependent chemiluminescence method by stimulation of formyl-methionyl-leucyl-phenylalanine. Oxidative free radical generating capacity of polymorphonuclear cells in patients with active ulcerative colitis was markedly enhanced as compared with control (p less than 0.01), while this enhanced free radical production by the cells was not detected at remission stage. Serial analyses revealed that oxidative free radical production by the cells in patients with ulcerative colitis was markedly enhanced with clinical deterioration of the disease, but it returned to normal level with an improvement of clinical features. These results suggest that the increased oxidative free radical production by polymorphonuclear cells could be related to the pathogenesis or aggravation of ulcerative colitis.

Role of macrophages in regeneration of liver.

In an attempt to clarify the role of macrophages and their mediators during regeneration of the liver, the difference of liver regeneration among C3H/HeN (LPS-responsive strain) and C3H/HeJ (LPS-resistant strain) mice was investigated. After a 67% partial hepatectomy, an increase in the weight of regenerating liver was significantly delayed in the C3H/HeJ mice, as compared with C3H/HeN mice. The number of hepatocytes labeled with antibody against PCNA reached maximum levels 48 hr after partial hepatectomy, but the PCNA labeling index in C3H/HeJ mice was 20% less than that for C3H/HeN mice. In addition, TNF-alpha activities in serum were enhanced shortly after partial hepatectomy in C3H/HeN strain mice, but were not increased in C3H/HeJ strain mice. Serum IL-6 levels were markedly enhanced in both C3H/HeN and C3H/HeJ mice, but a bimodial peak (14 and 48 hr after partial hepatectomy) was demonstrated in C3H/HeN mice, in contrast to a single peak (at 24 hr) in C3H/HeJ mice. Suppression of Kupffer cells by previous administration of gadolinium chloride in C3H/HeN mice reduced the increase in both serum TNF-alpha and IL-6 concentrations, reduced PCNA labeling index of hepatocytes by 20%, and disturbed the regeneration of the liver. Previous administration of antibody against TNF-alpha reduced the PCNA labeling index of hepatocytes by 20% after partial hepatectomy in C3H/HeN strain mice. These results suggest that LPS-responsive macrophages in the liver and their mediators, especially TNF-alpha, could partly play a role in liver regeneration.