Adjacent Hepatocytes Research Articles

Transmission electron microscopic study of hepatocytes in bioartificial liver.

A bioartificial liver (BAL) was prepared by simple inoculation of hepatocytes into the inner space of hollow fibers of a hemodialyzer and it was maintained in a closed circuit for in vitro culture. Morphology of hepatocytes in the hollow fibers was studied in detail using transmission electron microscopy (TEM). The hepatocytes formed three-dimensional, rod-shaped aggregates of 200 microm in diameter throughout the whole dimension of the hollow fibers after 1 day of culture. Approximately five hepatocyte layers existed from the surface to the center of the aggregate. The hepatocytes in the aggregate displayed mostly polygonal shapes and were surrounded by five to six cells. Abundant bile canaliculi were formed between the hepatocytes and were sealed by tight junctions. The distance between the adjacent hepatocytes except the bile canaliculus domain was approximately 20 nm, and interdigitation was observed between some hepatocytes. These observations indicate that the hepatocytes formed functionally associated aggregates, that is, organoids. Although the cells facing the inner surface of the hollow fiber lost their polygonal shape and became flattened during the following several-day culture, no drastic change was observed in the morphology of the hepatocytes located inside the aggregate. After 14 days of culture, the number of living cells decreased and most of these had a deformed nucleus, few numbers of organelles, and intermittent lipid droplets.

Transplanted reporter cells help in defining onset of hepatocyte proliferation during the life of F344 rats.

Transplanted hepatocytes integrate in the liver parenchyma and exhibit gene expression patterns that are similar to adjacent host hepatocytes. To determine the fate of genetically marked hepatocytes in the context of hepatocellular proliferation throughout the rodent life span, we transplanted Fischer 344 (F344) rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. The proliferative activity in transplanted hepatocytes was studied in animals ranging in age from a few days to 2 yr. Transplanted hepatocytes proliferated during liver development between 1 and 6 wk of age, each dividing an estimated two to five times. DNA synthesis in occasional cells was demonstrated by localizing bromodeoxyuridine incorporation. There was no evidence for transplanted cell proliferation between 6 wk and 1 yr of age. Subsequently, transplanted cells proliferated again, with increased sizes of transplanted cell clusters at 18 and 24 mo of age. The proliferative activity of transplanted cells was greater in rats entering senescence compared with during postnatal liver development. In old rats, some liver lobules were composed entirely of transplanted cells. We conclude that hepatocyte proliferation in the livers of very young and old F344 rats is regulated in a temporally determined, biphasic manner. The findings will be relevant to mechanisms concerning liver development, senescence, and oncogenesis, as well as to cell and gene therapy.

Suppression by Chai- hu- gui- zhi- tang of the development of liver lesions induced by N-nitrosomorpholine in Sprague–Dawley rats

The effects of Chai- hu- gui- zhi- tang (TJ-10) on the development of liver lesions induced by N-nitrosomorpholine and on labeling and apoptotic indices were investigated in male Sprague–Dawley rats. Rats were given chow pellets containing 0.5 or 1.0% TJ-10 and, from the beginning of the experiment, were given drinking water containing N-nitrosomorpholine for 8 weeks. Visible white liver nodules, cellular alteration foci, and hepatic foci staining positively for glutathione- S-transferase, placental type, were examined macroscopically or histochemically. In week 16, TJ-10 at both dosages significantly reduced the incidence, and/or number of visible white nodules and hepatic lesions. Quantitative histologic analysis also showed that prolonged feeding of TJ-10 at both dosages significantly reduced the number of hepatic foci positive for glutathione- S-transferase, placental type. Administration of TJ-10 at both dosages also significantly decreased the labeling index of adjacent liver and significantly increased the apoptotic index of adjacent liver but had no significant effect on those of neoplastic lesions. These findings indicate that TJ-10 suppresses the development of liver lesions and suggest that this effect might be related to TJ-10’s inhibition of cell proliferation and induction of apoptosis in adjacent hepatocytes.

Lessons From Genetically Engineered Animal Models VI. Liver repopulation systems and study of pathophysiological mechanisms in animals.

The ability to localize transplanted hepatocytes in the liver offers exciting new opportunities. Transplanted hepatocytes enter liver plates, form hybrid plasma membrane structures with adjacent hepatocytes, express liver genes correctly, and survive indefinitely. The transplanted cell mass is regulated, such that cell proliferation is limited in the normal adult liver, whereas the liver is repopulated extensively when proliferation rates in transplanted and host hepatocytes become dissociated or host hepatocytes are ablated selectively. Transplanted hepatocytes are susceptible to hepatitis viruses. These aspects of transplanted hepatocyte biology indicate that liver repopulation systems can help address questions concerning pathophysiological mechanisms.

Modulation of paracetamol metabolism by Kupffer cells: A study on rat liver slices

Recent studies support the hypothesis that non parenchymal cells (mainly macrophages) may play a role in the metabolism and cellular effects of paracetamol. In order to investigate this hypothesis, male Wistar rats were intravenously injected with either 7.5 mg kg gadolinium chloride (Gd+) or NaCl 0.9% (Gd−). The treatment with GdCl 3 decreased the number and the function of Kupffer cells in liver tissue, as assessed by the histological examination of the liver after colloidal carbon injection in the portal vein. Precision-cut liver slices (PCLS) were prepared from both groups of rats and cultured for 8h in Waymouth's medium in the presence and absence of 5 mM paracetamol. Interestingly, PCLS obtained from Gd+ rats exhibited a lower release of tumor necrosis factor (TNF-α) and a better viability than PCLS from control (Gd−) rats. Incubation with paracetamol led to a decreased glycogen level in liver slices from Gd+ or Gd−, without modifying neither liver morphology nor ATP level nor LDH release. A higher proportion of paracetamol glucuronide, was secreted from the slices obtained from Gd+ rats. These data suggest that Kupffer cells could affect the viability of PCLS in culture and are involved in the regulation of phase II metabolism in the adjacent hepatocytes. We propose that PCLS in culture is a suitable model to elucidate the biochemical mechanism underlying the modulation of metabolism occurring through hepatocytes-Kupffer cells interactions.

Phenotypical and morphological alterations to rat sinusoidal endothelial cells in arterialized livers after portal branch ligation.

The hepatic sinusoids are preferentially supplied with portal venous blood and equipped with fenestrated endothelial cells that are distinct from capillary endothelial cells. We previously observed in rats that sinusoidal capillarization proceeded concurrently with arterial blood supply during hepatocarcinogenesis. This study aimed to clarify the inducing role of arterialization in sinusoidal capillarization by investigating phenotypical, morphological and functional alterations to sinusoidal endothelial cells (SECs) in arterialized rat livers induced by portal branch ligation. At one week, after massive hepatic necrosis following ligation, the livers were restored to their normal architecture without causing post-necrotic fibrosis. At 12-21 weeks, they exhibited a normal histology except for mild pericellular fibrosis which developed along sinusoids or between adjacent hepatocytes. SECs expressed factor VIII-related antigen and showed a decrease in the number of fenestrae and porosity, still lacking any basement membrane but further retaining the functional capacity for carmine dye uptake. Stellate cells, while occasionally associated with large amounts of collagen bundles, contained many lipid droplets and expressed no alpha-smooth muscle actin, indicating a quiescent property. Kupffer cells were commonly found within the sinusoids. The present results indicate that arterialization of the liver induces a partial (but not complete) transition of SECs into capillary-type endothelial cells, suggesting that arterialization might be one of the factors which induce sinusoidal capillarization in the development of hepatocellular carcinoma.

Evidence of Hepatocyte Apoptosis in Rat Liver after the Administration of Carbon Tetrachloride

In acute liver injury induced by the injection of CCl4, cell death has been attributed to the necrosis of hepatocytes in the centrilobular area. In the present study, we re-examined the hepatic injury evoked by CCl4 in rats and explored the possibility that apoptosis may also contribute to its pathogenesis. Apoptotic hepatocytes were identified and quantified by light and electron microscopy, the in situ immunohistochemical labeling of nuclear DNA fragmentation, flow cytometry, and DNA gel electrophoresis. We found that a substantial number of hepatocytes underwent apoptosis. Apoptotic changes were also observed in ballooned hepatocytes. Apoptotic hepatocytes increased in number at 3 hours and peaked at 6 hours after the CCl4 injection. Apoptotic bodies were sequestrated in the adjacent hepatocytes and sinusoidal cells. Double staining of the cells with immunostaining for phagocytes and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining for labeling of DNA fragmentation showed that the majority of apoptotic hepatocytes were phagocytosed by Kupffer cells and macrophages. The results indicated that apoptosis occurs in the ballooned and injured hepatocytes of the centrilobular area. What occurs after CCl4 administration may be important in reducing inflammation, shortening the course of acute hepatic injury, and preventing the development of fibrosis.

Localization of Gamma-Glutamyltranspeptidase and Alkaline Phosphatase in Primary Cultures of Fetal Rat Hepatocytes.

The localization of gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (ALP) in primary cultures of fetal rat hepatocytes was investigated using enzyme cytochemical and immunocytochemical techniques. When hepatocytes were cultured in a medium supplemented with dexamethasone (DEX) or dimethylsulfoxide (DMSO), both enzymes were observed in the plasma membranes along the intercellular spaces that developed between adjacent hepatocytes. On the other hand, in hepatocytes cultured in the basic medium or in medium supplemented with transforming growth factor-β (TGF-β), the enzymes were found in the limited area of the cytoplasm surrounding the nuclei. Connexin-32, a subunit of proteins that comprise gap junctions, was detected along the cell borders in hepatocytes cultured in DEX or DMSO-supplemented medium, but was not found in hepatocytes cultured in the basic medium or in medium supplemented with TGF-β. However, the DEX and DMSO supplements did not cause any changes in the distribution of microtubules in hepatocytes, and numerous microtubular fibers were observed to extend in a radial pattern from the nuclei to the periphery of the cytoplasm in all hepatocytes, irrespective of medium type. These results indicate that assessment of the location of GGT and ALP may help determine the level of hepatocyte differentiation in cultured rat hepatocytes and that intracellular factors other than microtubules are involved in the transport of these enzymes from the cytoplasm to the plasma membrane.

Liver stem cells: when the going gets tough they get going.

The ability of the liver to regenerate is widely acknowledged, and this is usually accomplished by the entry of normally proliferatively quiescent hepatocytes into the cell cycle. However, when hepatocyte regeneration is impaired, small bile ducts proliferate and invade into the adjacent hepatocyte parenchyma. In humans and experimental animals these ductal cells are referred to as oval cells, and their association with defective regeneration has led to the belief that they are the progeny of facultative stem cells. Oval cells are of great biological interest since they may represent a target population for hepatic carcinogens, and they may also be useful vehicles for ex vivo gene therapy for the correction of inborn errors of metabolism. The ability of oval cells to differentiate into hepatocytes has been demonstrated unequivocally. However, this process only occurs when the regenerative capacity of hepatocytes is overwhelmed, and thus, unlike the intestinal epithelium, the liver is not behaving as a classical continually renewing stem cell-fed lineage.

Chemoprevention by amiloride against experimental hepatocarcinogenesis induced by N-nitrosomorpholine in Sprague-Dawley rats

The effects of prolonged administration of the diuretic amiloride on hepatocarcinogenesis induced by N-nitrosomorpholine (NNM) and the labeling index of the liver were investigated in male Sprague-Dawley rats. Rats were given drinking water containing NNM for 8 weeks and received s.c. injections of 2.5 mg/kg or 5.0 mg/kg body weight of amiloride every other day until the end of the experiment at week 16. Preneoplastic and neoplastic lesions staining positively for glutathione-Stransferase, placental type (GST-P) were examined immunohistochemically. In week 16, administration of amiloride at 2.5 mg/kg body weight significantly reduced the size (as mean area) of GST-P-positive lesions, and amiloride at 5.0 mg/kg body weight significantly reduced the number (as no./cm 2) and sizes (as mean area and percent of parenchyma) of GST-P-positive lesions. Amiloride at both dosages also significantly decreased the labeling index of enzyme-altered lesions and amiloride at higher dosage significantly decreased the labeling index of adjacent hepatocytes. These findings indicate that amiloride inhibits hepatocarcinogenesis and suggest that this effect may be closely related to amiloride's inhibition of cell proliferation in enzyme-altered lesions and/or adjacent liver.

Enhancement by ethyl alcohol of experimental hepatocarcinogenesis induced by N-nitrosomorpholine.

The effects of ethyl alcohol (EtOH) during or after treatment with N-nitrosomorpholine (NNM) on hepatocarcinogenesis, ornithine decarboxylase (ODC) activity and the labeling index of the liver were investigated in male Sprague-Dawley rats. Rats were given drinking water containing NNM for 8 weeks and received i.p. injections of 1 g EtOH/kg body weight every other day during or after treatment with NNM. Pre-neoplastic and neoplastic lesions staining positively for glutathione-S-transferase, placental type (GST-P), were examined immunohistochemically. At the end of experiment at week 16, administration of EtOH after NNM treatment had no significant effect on the number and size of GST-P-positive hepatic lesions, whereas administration of EtOH during NNM treatment significantly increased the number and percentage area but not the mean area of GST-P-positive hepatic lesions. EtOH caused significant increases in the ODC activity of the liver and in the labeling indices of enzyme-altered lesions and the adjacent hepatocytes after the cessation of EtOH administration but not during EtOH treatment. Our findings indicate that EtOH enhances hepatocarcinogenesis and suggest that this effect may be closely related to the increases in ODC activity and cell proliferation in enzyme-altered lesions and the adjacent liver after EtOH treatment.

Hepatic overexpression of MHC Class II antigens and macrophage-associated antigens (CD68) in patients with biliary atresia of poor prognosis

The pathogenesis of biliary atresia (BA) is still unknown. Progression to cirrhosis despite restoration of bile flow by successful portoenterostomy suggests that it is a progressive disease of the liver and biliary tree. Whether immunologic factors play any role in the development of this disease remains uncertain. Aberrant expression of major histocompatibility complex (MHC) Class II antigens of HLA-DR on hepatocytes and biliary epithelium is regarded as important in the progression of hepatocellular and biliary damage mediated by cytotoxic T cells. This study was undertaken to evaluate expression of MHC Class II antigen and macrophage-associated antigens (CD68) in liver of patients with biliary atresia to determine their prognostic usefulness and possible role in the pathogenesis of the disease. Liver biopsy specimens from infants with BA (n = 15), neonatal hepatitis (n = 3), and normal livers (n = 6) were studied using an indirect immunoperoxidase staining using antibodies against MHC Class II antigen and macrophage-associated antigens (CD68) as well as routine H&E and Masson's trichome stain. In patients with biliary atresia, the liver biopsy specimen was obtained at the time of Kasai portoenterostomy. Expression of HLA-DR antigens and CD68 antigens was either absent or minimal in normal liver biopsy specimens. There were a few HLA-DR antigens and a few CD68-positive cells around portal tracts in all patients with neonatal hepatitis and five of the seven biliary atresia patients with successful Kasai portoenterostomy. In contrast, there was strong expression of HLA-DR antigen in bile ductules, inflammatory cells, and adjacent damaged hepatocytes and marked CD68-positive macrophage infiltrate in the portal tracts as well as hepatic lobules in two patients with good prognosis and in all eight patients with bad prognosis. Hepatic expression of MHC Class II antigen and CD68 antigens correlated well with the severity of clinical course in patients with BA and may act as a prognostic factor in these patients.

Hepatic canalicular membrane 1: The role of mdr2 P-glycoprotein in hepatobiliary lipid transport.

The small apical (canalicular) domains of hepatocytes form a luminal meshwork of tubules between adjacent hepatocytes and are the sites of primary bile formation. Organic compounds are transported across this membrane domain against high concentration gradients. It has been recognized in recent years that the hepatocyte is harnessed with a set of canalicular ATP-dependent transport proteins, specialized in this uphill transport. Bile salts, organic anions, cations, and neutral amphipaths are all pumped into the bile via such primary active transporters. Functionally, these transporters resemble ABC transporters overexpressed in cells with the multidrug resistance phenotype. Indeed, those transporters that have been characterized at the molecular level turn out to be new, or already recognized, members of this family. Phospholipid secretion across the canalicular membrane of the mouse is also mediated by a member of this family, mdr2 P-glycoprotein. This was demonstrated by the absence of phospholipid secretion into bile of mice with a disrupted mdr2 gene and by subsequent demonstration of phospholipid translocation in cells that overexpress this protein. The recognition of mdr2 P-glycoprotein as a phospholipid flippase sheds new light on the function of P-glycoproteins and is an important step in understanding the mechanism of biliary lipid secretion.

Inhibition by the calmodulin antagonist trifluoperazine of experimental hepatocarcinogenesis induced by N-nitrosomorpholine in Sprague-Dawley rats.

The effect of the calmodulin antagonist trifluoperazine on hepatocarcinogenesis induced by N-nitrosomorpholine (NNM) and on the ornithine decarboxylase (ODC) activity and labeling index of the liver were investigated in male Sprague-Dawley rats. Rats were given drinking water containing NNM for 8 weeks, and from the beginning of the experiment, received s.c. injections of 15 or 30 mg/kg body weight of trifluoperazine in depot form every other day until the end of the experiment. Preneoplastic and neoplastic lesions staining positively for glutathione-S-transferase, placental type (GST-P) were examined histochemically. In week 16, quantitative histological analysis showed that prolonged administration of 30 mg but not 15 mg/kg body weight of trifluoperazine resulted in significant reductions in the number and percentage area of GST-P-positive hepatic lesions. Trifluoperazine also caused significant decreases in the ODC activity of the liver and in the labeling indices of enzyme-altered lesions and their adjacent hepatocytes. These findings indicate that trifluoperazine inhibits carcinogenesis and suggest that this effect may be closely related to its effect in inhibiting ODC activity and cell proliferation in the enzyme-altered lesions and their adjacent liver.

The Lesions of Hepatic Fatty Cirrhosis in Sheep

During a natural outbreak of hepatic fatty cirrhosis (HFC) in western Texas, 500 2-6-year-old Rambouillet ewe sheep were sequentially studied to determine the pattern of lesion development. All sheep developed lesions of HFC. Grossly, changes first began in the subcapsular hepatic parenchyma along the porta hepatis and spread peripherally until, in the final stages of the disease, approximately 80% of the liver was affected. Ascites, hydropericardium, and acquired hepatic vascular shunts were present in sheep with severe HFC. Light microscopic lesions initially appeared as accumulations of fine lipid droplets in the cytoplasm of periacinar hepatocytes but, with time, involved all hepatocytes of the lobule. The fat vacuoles in the periacinar hepatocytes coalesced to form larger vacuoles; and after rupture of adjacent fat-laden hepatocytes, fatty cysts appeared. Fibrosis began in the periacinar zone associated with the ruptured fatty cysts and continued until there was widespread bridging periacinar fibrosis. Islands of regenerating hepatocytes were frequently sequestered within the bands of fibrous tissue. Characteristically, the hepatic and posterior mediastinal lymph nodes, lung, and spleen contain ceroid. No lesions of hepatic encephalopathy were found in any animal. HFC is a progressive, chronic disease of sheep, and the morphology of the hepatic lesions is similar to lipotrope-deficient forms of nutritional cirrhosis. These findings are discussed in relation to similar nutritional deficiencies and toxicoses in sheep.

ICAM-1 Expression in Hepatocytes Following Dissociation of Cell-to-Cell Contact in Rats

ICAM-1 was not detected immunohistologically in hepatocytes in normal rats, but detectable in centrilobular degenerative hepatocytes in carbon tetrachloride-intoxicated rats. ICAM-1 expression was observed even in normal hepatocytes following liver perfusion with Hank′s balanced salt solution at a flow rate of 4.2 mL/g liver weight/min or with the same solution containing collagenase or EGTA at the physiological flow rate (1.4 mL/g liver weight/min). Such expression was also observed when liver perfusion was performed after pretreatment of rats with cycloheximide. On electron microscopy, ICAM-1 was exclusively stained on hepatocyte plasma membrane that was detached from the plasma membrane of adjacent hepatocytes. ICAM-1 mRNA and ICAM-1 protein were detected in hepatocytes freshly isolated from normal rats. Thus, ICAM-1 expression in degenerative hepatocytes as well as in hepatocytes following liver perfusion can be assumed to result from dissociation of cell-to-cell contact.

Enzyme cytochemical study of alkaline phosphatase in rat hepatocytes--change of location after colchicine administration or bile duct ligation.

Changes in location of alkaline phosphatase (ALP) in rat hepatocytes after two different treatments were examined by the enzyme cytochemical technique. Although reaction deposits which indicate ALP activity were observed mainly in the bile canalicular membranes until 6 hr after colchicine administration, they were present not only in the bile canalicular membranes but also in the sinusoidal and lateral membranes 12 hr after drug administration. Eighteen and 24 hr after colchicine administration, a large number of reaction deposits were seen in the sinusoidal membranes and in the mutual membranes between adjacent hepatocytes, where the bile canaliculi were completely deformed and complex interdigitations appeared. On the other hand, after bile duct ligation, reaction deposits appeared in every domain of the plasma membranes. Twenty-four hr after colchicine administration to bile-duct-ligated rats, an increase in the number of the reaction deposits was observed, but the location of ALP remained unchanged. The present study suggests that intracellular transport and location of ALP in hepatocytes may be controlled by a common mechanism in colchicine-treated and bile-duct-ligated rats.

Liver of juvenile Atlantic salmon, Salmo salar L.: a light, transmission, and scanning electron microscopic study, with special reference to the sinusoid.

This report provides a detailed description of sinusoidal and perisinusoidal structures in the normal liver of the juvenile Atlantic salmon (Salmo salar L.), a teleost species. The liver was studied by light, transmission, and scanning electron microscopy, and organ specimens were sampled after retrograde, whole-body perfusion through the dorsal aorta using 3% glutaraldehyde. Detailed characterization of perisinusoidal stellate cells also included immunohistochemical staining for desmin and evaluation of autofluorescence of the same cells upon excitation in ultraviolet (UV) light. The sinusoid is lined by one cell type only: the endothelial cell. No intraluminal pit cells or Kupffer cells are present. The space of Disse contains reticulin fibres, visualized by Gomori's silver stain, and perisinusoidal stellate cells (PSC). PSC exhibited autofluorescence in UV light, indicating that these cells store vitamin A in cytoplasmic lipid droplets. Immunohistochemically, PSC were found negative for desmin. The space of Disse, extending deep down between adjacent hepatocytes, receives long, slender microvilli from parenchymal cells. In addition to scattered macrophages, interhepatocytic cells (IHC) are found perisinusoidally. Hepatocytes of Atlantic salmon form branching and anastomosing tubules. The sinusoids of Atlantic salmon liver are lined by a fenestrated endothelium, with PSC located in the space of Disse, with macrophages and IHC as inhabitants of the interhepatocytic space. IHC show ultrastructural similarities to mammalian pit cells and teleostean large granular lymphocytes, as well as to piscine monocytes. PSC might be storage cells for vitamin A in Atlantic salmon as shown by autofluorescence in these cells, while immunohistochemical studies indicate that desmin does not seem to be an adequate immunohistochemical marker for PSC in the juvenile Atlantic salmon. Methodologically, fixation for electron microscopy was performed by a new and convenient perfusion method: arterial retrograde whole body perfusion. Liver specimens intended for scanning electron microscopy were fractured at room temperature after prolonged osmium postfixation, leaving hepatocytes intact and producing images well suited to document the three-dimensional structure of cells and tissue.

Gap Junction-Mediated Intercellular Communication in Primary Cultures of Rainbow Trout Hepatocytes

Gap junction-mediated intercellular communication was evaluated in primary cultures of rainbow trout hepatocytes by measurement of dye coupling. Donor hepatocytes were microinjected with fluorescent Lucifer yellow CH dye and visualization of dye spread (dye coupling) to adjacent hepatocytes was recorded. A maximum level was reached between 8 and 12 hr which was maintained up to 24 hr. Dye coupling then decreased over the next 48 hr. Cell viability was monitored by the percentage of total LDH released and trypan blue exclusion at each time point. The effect of 12- O-tetradecanoylphorbol-13-acetate (TPA), a known inhibitor of rodent hepatic intercellular communication, on gap junction-mediated intercellular communication was evaluated in 24-hr cultures. A dose-response inhibition was demonstrated with maximum inhibition observed at 3 hr of exposure.

Depletion of gamma interferon and tumor necrosis factor alpha in mice with Rickettsia conorii-infected endothelium: impairment of rickettsicidal nitric oxide production resulting in fatal, overwhelming rickettsial disease.

C3H/HeN mice infected intravenously with a dose of Rickettsia conorii (Malish 7 strain) that is sublethal for immunocompetent animals (1.1 x 10(3) PFU) developed disseminated infection of endothelial cells of the brain, lungs, heart, liver, kidney, testis, and testicular adnexa. In R. conorii-infected mice depleted of gamma interferon (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) by intravenous administration of neutralizing monoclonal antibodies on days 0, 2, and 4, the mortality rate was 100%. Death of the cytokine-depleted animals on days 5 and 6 was associated with overwhelming rickettsial infection documented by titration of rickettsial content in the brain and liver and by immunohistologic demonstration of massive quantities of R. conorii in endothelial cells of all organs examined, in macrophages of the liver and spleen, and in hepatocytes. Nondepleted, immunocompetent animals showed markedly reduced rickettsial content in the tissues on day 6, with rickettsial destruction in phagolysosomes not only in macrophages but also in endothelial cells and hepatocytes. All nondepleted, infected mice recovered and appeared completely healthy by day 9. Assay of liver infiltrated by lymphocytes and macrophages revealed mRNA of IFN-gamma and TNF-alpha, indicating that the host defenses were activated at the site of infection. Treatment of mice with an analog of L-arginine reduced the synthesis of nitric oxide and impaired rickettsial killing. Nitric oxide production was also impaired in cytokine-depleted infected mice. These observations support the hypothesis that IFN-gamma secreted by T lymphocytes and natural killer cells and TNF-alpha secreted by macrophages act in a synergistic, paracrine fashion on adjacent rickettsia-infected endothelial cells, hepatocytes, and macrophages to stimulate synthesis of nitric oxide, which kills intracellular R. conorii.