A study on renal papillary necrosis experimentally produced by the Shwartzman mechanism in rabbits

To produce renal papillary necrosis experimentally by means of the Shwartzman mechanism in rabbits, E. coli endotoxin was injected into the renal pelvis unilaterally through the ureter as a preparative procedure after pretreatment by local administration of alcohol, and the same endotoxin was given again 24 hours later, but intravenously this time via the ear vein, as a provocation. Marked necrosis was produced in the renal papillae, where many intravascular fibrin thrombi were found histologically. Such papillary necrosis was largely prevented by heparin administration, and this lesion was considered to be the univisceral Shwartzman reaction occurring in the renal papillae. The lesion produced in the new experimental system of renal papillary necrosis described here has a good similarity to that of human cases in etiology, pathogenesis and morphology. The present system may therefore be a good model of human renal papillary necrosis, and should be useful for future studies.     

Acute,massive, haemorrhagic adrenal necrosis experimentally produced by the Shwartzman mechanism in rabbits

Summary Acute and severe haemorrhagic necrosis of the adrenal was produced experimentally in rabbits by means of intravenous injection of endotoxin after pretreatment by adrenocorticotropic hormone (ACTH) administration. The change occurred mainly in thezona fasciculata of the adrenal cortex, and its pathology was quite similar to that of the Shwartzman reaction. Numerous microthrombi were found in and around the lesion, but no marked changes were seen in other parts of the body. Heparin administration was very effective in preventing the necrosis. The pathogenesis of this lesion was postulated to be a univisceral Shwartzman mechanism in the adrenal. This seems to be a good experimental model for massive haemorrhagic necrosis of the adrenal in man, for example in the Waterhouse-Friderichsen syndrome, the pathogenesis of which has been assumed to involve intravascular clotting. It is suggested that hyperfunction of the adrenal cortex caused by ACTH administration could be a preparative condition for the Shwartzman reaction.This study was supported in part by a grant-in-aid from the Japanese Ministry of Education, Science and Culture     

Mast cell density during initiation and progression of the local Shwartzman reaction

Objective and design: To quantify the number of mast cells in the skin of rabbits during initiation and progression of the local Shwartzman reaction.Materials: Thirty New Zealand rabbits were divided in two groups (n = 15/group). One group was subjected to the Shwartzman reaction and the other group served as control. Subsequently, animals were further subdivided in six groups of five animals each according to time of euthanasia.Treatments: The local Shwartzman reaction was induced by two inoculations of Salmonella typhimurium lipopolysaccharide. Preparatory inoculation was given intradermally and, 24 h later, the provocative injection was administered intravenously. Controls were subjected to the same procedure but received saline. After provocative injection animals were killed at 1, 8, and 15 days.Methods: Skin samples were fixed in Carnoys solution and mast cells were identified employing a low pH toluidine blue stain. Numbers of mast cells were determined per square millimetre and, subsequently, those cells degranulated were identified and quantified to obtain absolute values. A Students t test was initially used to compare Shwartzman versus controls at each time point. Subsequently, an ANOVA test employing a factorial experiment was used to assess a possible interaction between time of euthanasia and treatments.Results: The values were transformed (natural logarithms) for appropriate statistical comparisons. Independent comparisons at each time point showed that Shwartzman groups had higher numbers of mast cells than controls at 1 and 8 days, but not at 15 days (5.71 ± 1.00 Vs. 2.40 ± 0.58, P < 0.005; 3.77 ± 0.90 Vs. 2.33 ± 0.56, P < 0. 025, and 2.61 ± 0.25 Vs. 2.39 ± 0.39, P > 0.05, respectively). Degranulated cells were numerous in Shwartzman groups, particularly at day 1 (3.48 ± 0.78) and less obvious at day 8 (0.72 ± 0.50), but were scarce by day 15 (–0.67 ± 0.99) as well as controls (–0.68 ± 0.91). The factorial experiment demonstrated that the Shwartzman reaction and time of euthanasia were independently significant (P < 0.005) but their interaction at day 1 was the major contributor (P < 0.005). Tukeys w pairwise comparisons of means confirmed that the Shwartzman group killed at day 1 was significantly different from the others (P < 0.05).Conclusions: Mast cells significantly increase in the early stages of the local Shwartzman reaction. Thus, mast cells are a highly dynamic cell population, which have a prominent role during the acute phase of this lipopolysaccharide-induced inflammatory reaction but not during healing.Received 3 February 2003; returned for revision 5 March 2003; returned for final revision 29 September 2003; accepted by M. Parnham 4 November 2003     

Endotoxin induced hepatic necrosis in rats on an alcohol diet

The role of endotoxin in the pathogenesis of progressive liver disease is receiving increasing attention, but remains controversial. Similarly, although alcoholic hepatitis is now recognized as the transitional link between alcoholic fatty liver and advanced alcoholic liver disease, the aetiology of liver cell necrosis in alcoholic hepatitis is not known. Rats fed a nutritionally adequate liquid alcohol diet according to the formula of Lieber and DeCarli developed fatty livers. Littermates fed an identical diet and challenged with small IV doses (1 microgram/g body weight) of E. coli lipopolysaccharide endotoxin (LPS) developed focal necrotizing hepatitis. Control littermates fed an identical calorie balanced but alcohol free diet and challenged with identical doses of LPS did not develop any liver lesions. The hepatocyte necrosis with associated inflammatory changes induced by LPS in fatty livers has some features of early human alcoholic hepatitis and suggests that progressive alcohol induced damage may be multifactorial in origin.     

A model for the investigation of factors influencing haemorrhagic necrosis mediated by tumour necrosis factor in tissue sites primed with mycobacterial antigen preparations.

Mycobacterial lesions and skin sites challenged with soluble mycobacterial antigen are very sensitive to the necrotizing effect of tumour necrosis factor (TNF). We have used a model that permits separate quantitative assessment of swelling and haemorrhage to show that when these reactions are elicited in mice that have not been deliberately immunized, pretreatment of the mice with lipopolysaccharide (LPS), or with a MoAb to CR3 which blocks emigration of myeloid cells into the tissues, will block both the swelling and the haemorrhage. On the other hand, treatment with an inhibitor of platelet-activating factor (PAF), or with misoprostol (a synthetic prostaglandin E1 analogue), or with cobra venom factor (CVF) which depletes complement, preferentially blocks the haemorrhagic component, while leaving the swelling relatively unaltered. As swelling occurs before the haemorrhage is seen, it is possible that these factors act at a late stage in the cascade of events leading to the tissue damage. However, LPS and CVF were able to inhibit swelling and haemorrhage in the massive reactions elicited in pre-immunized animals, whereas the PAF inhibitor had no detectable effect.     

Electron microscopic observations of the kidney in the generalized Shwartzman reaction

Summary Sequential changes in the kidney during the generalized Shwartzman reaction were studied electron microscopically. The first anatomical change was infiltration of neutrophils into the glomerular capillaries. Endothelial damage was not noticeable until the capillaries were filled with fibrin deposits. Fibrin appeared in the mesangium at almost the same time as in the capillary lumina, traversing through the endothelial fenestrae. Endothelial damage was more common in the mesangial portion than in the peripheral portion of the capillaries. Severe mesangiolysis developed after loss of endothelial cells had been followed by massive penetration of intracapillary contents. Later, signs of repair were evident in some parts of the damaged endothelium. The development of cortical necrosis coincided with the appearance of mesangiolysis and arteriolar thrombotic lesions.Supported in part by grants from the Japanese Ministry of Education     

The diagnostic significance of periportal hepatic necrosis and inflammation

In this review the several types of cell damage and cell death which may be found in liver biopsy specimens are defined. We describe the different processes which occur at the portal/parenchymal or septal/parenchymal interface, viz. periportal spillover, periportal hepatitis, classic or lymphocytic piecemeal necrosis and biliary piecemeal necrosis. The diagnostic implications of these lesions in relation to the clinicopathological diagnosis and prognosis in various liver diseases are discussed.     

Acute hepatic cell necrosis experimentally produced by viral agents in rabbits.

Acute and extensive hepatic cell necrosis was produced experimentally in rabbits by means of the Shwartzman mechanism using adeno- and hepatitis B viruses. The change that occurred in the liver was quite severe, namely, areas of hemorrhagic necrosis of various sizes in gross appearance and lytic-coagulative necrosis with hemorrhage and leukocyte-mononuclear cell infiltration histologically. Thrombi formation was noted in and around the necrotic areas, and it was not unusual to see necrosis of an entire lobe. This seems to be a model, to some extent, for human fulminant hepatitis caused by hepatitis virus infection, and suggested that some nonspecific reaction such as intravascular clotting may also play an important role in causing or complicating acute, severe, and extensive necrosis of the liver in human cases. Heparin administration very effectively prevented such hepatic necrosis, which supports the view that the change we observed in the liver was really the Schwartzman reaction; further, it is reminiscent of the fact that heparin administration is sometimes effective in fulminant hepatitis treatment if given at te appropriate stage of the disease.     

Involvement of tumor necrosis factor-α, interleukin-I β, interleukin-8, and interleukin-I receptor antagonist in acute lung injury caused by local Shwartzman reaction

A local Shwartzman reactlon (LSR) was prepared in rabbit lung as a model of acute lung injury. To induce LSR, intratracheal injection of lipopolysaccharide (LPS) 10 μg into the lower lobe of the right lung, followed 24 h later by I.v. injectlon of LPS (10 μg/kg). In the lung with the LSR, myeloperoxidase activity, representing neutrophil accumulation, peaked at 1–2 h and was sustained for 48 h after challenge with i.v. LPS. The lung water content peaked at 12 h, and decreased gradually. Histological findings showed diffuse interstitial widenlng, lntra-alveolar leukocyte infiltration with hemorrhage, and alveolar exudate formation. The production of tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1β), interleukin 8 (IL-8), and IL-1 receptor antagonist (IL-1Ra) in the lung was analyzed. TNF-α first elevated and peaked at 0.5h (66.±16.7ng/g.lung), subsequently, 11–1β and 11–8 increased and peaked at 2h (17.8 ± 3.4 ng/g. lung and 336.9±49.6ng/g.lung, respectively). IL-1Ra was present even before the challenge, and the production increased to show a dual peak (0.5 h, 1.5±0.2 μg/g.lung; and 2h, 1.6±0.1 μg/g.lung), and a large concentration of IL-1 Ra was sustained for 48 h. lmmunohistochemistry showed that the cellular source of these cytokines was alveolar macrophages and infiltrating neutrophils. Thus, disclosing the kinetics of the generation of cytokines led to a better understanding of thelr roles, namely TNF-α as an initiator, IL-1 and IL-8 as amplitier and effector, and IL-1 Ra as regulator of the intensity of acute inflammation.     

Intragranulomatous necrosis in pulmonary granulomas is not related to resistance against Mycobacterium tuberculosis infection in experimental murine models induced by aerosol

Intragranulomatous necrosis is a primary feature in the natural history of human tuberculosis (TB). Unfortunately, this phenomenon is not usually seen in the experimental TB murine model. Artificial induction of this necrosis in pulmonary granulomas (INPG) may be achieved through aerosol inoculation of lipopolysaccharide (LPS) 3 weeks after Mycobacterium tuberculosis infection. At week 9 post-infection, the centre of primary granulomas became larger, showing eosinophilic necrosis. Interestingly, INPG induction was related to mice strains C57BL/6 and 129/Sv, but not to BALB/c and DBA/2. Furthermore, the same pattern was obtained with the induction of infection using a clinical M. tuberculosis strain (UTE 0335R) that naturally induces INPG. In all the mice strains tested, the study of pulmonary mRNA expression revealed a tendency to increase or to maintain the expression of RANTES, interferon-gamma, tumour necrosis factor and iNOS, in both LPS- and UTE 0335R-induced INPG, thus suggesting that this response must be necessary but not sufficient for inducing INPG. Our work supports that INPG induction is a local phenomenon unrelated to the resistant (C57BL/6 and BALB/c) or susceptible (129/Sv and DBA/2) background of mice strains against M. tuberculosis infection.     

Curcuma longa extracts suppress pathophysiology of experimental hepatic parenchymal cell necrosis

The study sought to investigate the protective potentials of Curcuma longa rhizome following potassium bromate-induced liver injury in Wistar rats. Thirty-five male Wistar rats were divided into 7 groups of 5 rats each (n = 5). Control group received normal saline while the other groups received oral administration of 100 mg/kg potassium bromate daily for two weeks to induce hepatic injury. Negative control I rats were sacrificed immediately after induction of hepatic injury, while the test groups were given oral dose of ethanol extract of Curcuma longa rhizome (EECLOR) at 100, 200 and 400 mg/kg for two weeks. Positive control group was treated with Silymarin for two weeks, while negative control II group was observed for the two-week period. At the end of the study, serum biochemical parameters of liver function enzymes, malondialdehyde and histopathological changes were investigated. Necrotic hepatocytes were quantified in H&E-stained liver sections using the morphologic criteria of typical necrotic tissue. Hepatocytes that remained intact were identified as those with round euchromatic nuclei with prominent nucleoli. Histological examination and morphological grading of the stained sections showed massive necrosis across the zones. EECLOR improved liver functions evidenced by reduced activity of serum amino transferases. It also reduced lipid peroxidation. In addition, there was significant reduction of hepatocytes showing morphological criteria of necrosis in EECLOR-treated rats across the zones, with appreciable radial sinusoidal arrangement. In conclusion, the protective actions of EECLOR against potassium bromate liver toxicity in rats, appears to be due to its ability to reduce lipid peroxidation.     

On the mechanism of glucagon stimulation of hepatic gluconeogenesis

Summary The addition of L-alanine as substrate to a perfused rat liver preparation produced a five-fold increase in the rate of glucose production. This enhancement of the gluconeogenic flux seems to be a consequence of a rise in the steady-state levels of pyruvate and oxaloacetate subsequent to the rise in alanine concentration.Glucagon (2×10^–9 M) increased the gluconeogenic flux from alanine (10 mM) by 50 percent, even though the concentration of the substrate in the perfusion fluid was at saturation. This effect was accompanied by a rise in the intracellular concentrations of alanine. However, the steady-state concentrations of pyruvate and oxaloacetate were decreased, probably as a consequence of a more reduced state of the nicotinamide-nucleotide system. In vivo, the intraperitoneal administration of glucagon to starved rats was accompanied by a decrease in the hepatic alanine and pyruvate concentrations despite the striking effects raising the plasma glucose levels. These observations seem to indicate that the effect of the hormone increasing the hepatic glucose output must be mediated through some other mechanism(s) independent of the intracellular variations in the hepatic amino acids levels.     

Vascularization in tissue remodeling after rat hepatic necrosis induced by dimethylnitrosamine

We observed postnecrotic tissue remodeling to examine vascularization in adult rat livers. Livers, bone marrow, and peripheral blood from rats at 24 h to 14 days after an injection of dimethylnitrosamine (DMN) were examined by light microscopic, immunohistochemical, and ultrastructural methods. Numerous ED-1 (a marker for rat monocytes/macrophages)-positive round mononuclear cells infiltrated in the necrotic areas at 36 h after DMN treatment. On day 5, when necrotic tissues were removed, some of the cells were transformed from round to spindle in shape. On day 7, these cells were contacted with residual reticulin fibers and became positive for SE-1, a marker of hepatic sinusoidal endothelial cells and Tie-1, an endothelial cell-specific surface receptor, associated with frequent occurrence of ED-1/SE-1 and ED-1/Tie-1 double-positive spindle cells. Ultrastructurally, the spindle cells simultaneously showed phagocytosis and endothelial cell-like morphology. With time necrotic areas diminished, and on day 14, the necrotic tissues were almost replaced by regenerated liver tissues and thin bundles of central-to-central bridging fibrosis. Bone marrow from 12 h to day 2 showed an increase of BrdU-positive mononuclear cells. Some of them were positive for ED-1. The BrdU-labeled and ED-1-positive cells appeared as early as 12 h after DMN injection and reached a peak in number at 36 h. They were similar in structure to ED-1-positive cells in necrotic liver tissues. These findings suggest that round mononuclear ED-1-positive cells proliferate first in bone marrow after DMN treatment, reach necrotic areas of the liver through the circulation, and differentiate to sinusoidal endothelial cells. Namely, hepatic sinusoids in DMN-induced necrotic areas may partly be reorganized possibly by vasculogenesis.     

The immunocytochemical localization of tumour necrosis factor and leukotriene in the rat heart and lung during endotoxin shock

After the intravenous administration of lipopolysaccharide at a dose of 3.0 mg/100 g to rats, immunoreactive sites for tumour necrosis factor (TNF) and peptide leukotrienes (LTs) were examined in the heart and lung. Immunoreaction for TNF is preferentially localized on the apical endothelial cell surface of the vessels and in lysosomes of inflammatory and interstitial cells. Lysosomes of cardiac muscle cells which undergo degeneration are also reactive. Peptide LTs in inflammatory cells give almost the same reactions as those for TNF. However, the production of peptide LTs occurs uniquely in cardiac muscle cells in the media of the pulmonary vein, although lysosomes of intracardiac muscle cells which undergo degeneration do not show immunoreactivity. These results suggest that the degeneration of cardiac muscle cells may be induced not only by endogenous TNF but also by peptide LTs which are produced in muscle cells of the venous media and are transported to the myocardium via the coronary circulation.     

IL-8 as a circulating cytokine: induction by recombinant tumour necrosis factor-alpha.

Tumour necrosis factor-alpha (TNF-alpha) is a pivotal cytokine at the centre of a cascade of cytokines and inflammatory mediators which modulate the host response to infection and trauma, and in particular the metabolic changes resulting in shock and subsequent multi-organ failure. The cytokine IL-8--predominantly an activator and chemotactic factor for circulating polymorphonuclear neutrophil leucocytes--is produced in response to TNF-alpha in vitro, and high circulating levels of IL-8 are found in septic primates. We have studied the release of IL-8 into the circulation of subjects with chronic hepatitis B undergoing a 10 week pilot trial of recombinant TNF-alpha (rTNF-alpha) therapy in doses of 15-100 micrograms/m2. A marked dose-dependent increase in plasma IL-8 levels was seen commencing at 30-60 min after the start of rTNF-alpha infusion and peaking between 2 and 3 h (mean peak level 4300 ng/l). The temporal pattern of IL-8 production exactly echoed that of IL-6, another component of the cytokine cascade, but peak plasma levels of IL-8 were up to 17 times higher than those of IL-6. This study confirms in vitro data suggesting that IL-8 is a component of the acute circulating cytokine cascade with a potential role in the modulation of the acute immune and metabolic response to infection and trauma.     

IgA1与系膜细胞共培养上清对足细胞分泌TNF-α的影响及机制探讨

目的： 探讨IgA肾病血清IgA1与系膜细胞共培养上清对足细胞分泌TNF-α的影响及机制。方法：Jacalin 亲和层析柱和Sephacryl S-200 分子筛用来纯化蛋白,单体IgA1（mIgA1)热聚合为聚合体IgA1(aIgA1), 同步化的系膜细胞与患者和健康对照来源的aIgA1共培养,收集上清,分别与同步化的足细胞作用;ELISA检测足细胞上清TNF-α水平,real time PCR 检测凋亡相关基因Ang、ACE mRNA表达情况。结果：IgAN患者来源的aIgA1与系膜细胞共培养得到上清可刺激足细胞分泌TNF-α,其水平显著高于对照组［(12.47±1.45) ng/L vs (2.33±0.65) ng/L,P<0.05］。与对照和健康上清组相比,该上清可上调足细胞Ang和ACE mRNA显著升高(P<0.05)。依那普利拉(10-5 mol/L)可使该上清作用后的足细胞分泌的TNF-α显著下降至(7.52±1.12) ng/L (P<0.05);而缬沙坦(10-5 mol/L)则使之下降至(6.64±0.68) ng/L (P<0.05);而依那普利拉(0.5×10-5 mol/L)和缬沙坦(0.5×10-5 mol/L)联合治疗可使足细胞TNF-α下降至(2.72±0.55) ng/L,与对照无显著差异(P>0.05)。结论：IgA肾病患者血清IgA1与系膜细胞共培养上清可通过激活肾素-血管紧张素系统而刺激足细胞TNF-α分泌,参与IgAN的进展。     

Fever produced in the rat by intracerebralE. coli endotoxin

Summary E. coli endotoxin introduced into the brain ventricles or into various brain areas raises the body temperature of conscious rats. Endotoxin-sensitive sites were found within the anterior hypothalamus and the lower brainstem. The increase in temperature (T) after microinjection of endotoxin into the anterior hypothalamic nucleus was dose-dependent. Endotoxin injected into the ventricles produced monophasic hyperthermia, but microinjections into the anterior hypothalamus produced monophasic or biphasic types of hyperthermia. The second and third microinjections of endotoxin into the anterior hypothalamic nucleus subsequently caused higher responses in T, but not in the rate of temperature change. The effects of the fourth and further microinjections were the same as those of the third, and no tolerance developed for 24 days. The observations of behaviour, vegetative reactions, skin temperature, and carbon dioxide production indicated that the rise in the rat body temperature induced by endotoxin represents fever and that the heat is gained, at an ambient temperature of 22°C, mainly through skin vessel vasoconstriction. Aspirin abolished and reversed fever induced by endotoxin, while hydrocortisone was without effect.     

CXCL10 promotes liver fibrosis by prevention of NK cell mediated hepatic stellate cell inactivation

Chemokines, such as CXCL10, promote hepatic inflammation in chronic or acute liver injury through recruitment of leukocytes to the liver parenchyma. The CXCL10 receptor CXCR3, which is expressed on a subset of leukocytes, plays an important part in Th1-dependent inflammatory responses. Here, we investigated the role of CXCL10 in chemically induced liver fibrosis. We used carbon tetrachloride (CCl(4)) to trigger chronic liver damage in wildtype C57BL/6 and CXCL10-deficient mice. Fibrosis severity was assessed by Sirius Red staining and intrahepatic leukocyte subsets were investigated by immunohistochemistry. We have further analyzed hepatic stellate cell (HSC) distribution and activation and investigated the effect of CXCL10 on HSC motility and proliferation. In order to demonstrate a possible therapeutic intervention strategy, we have examined the anti-fibrotic potential of a neutralizing anti-CXCL10 antibody. Upon CCl(4) administration, CXCL10-deficient mice showed massively reduced liver fibrosis, when compared to wildtype mice. CXCL10-deficient mice had less B- and T lymphocyte and dendritic cell infiltrations within the liver and the number and activity of HSCs was reduced. In contrast, natural killer (NK) cells were more abundant in CXCL10-deficient mice and granzyme B expression was increased in areas with high numbers of NK cells. Further detailed analysis revealed that HSCs express CXCR3, respond to CXCL10 and secrete CXCL10 when stimulated with IFNγ. Blockade of CXCL10 with a neutralizing antibody exhibited a significant anti-fibrotic effect. Our data suggest that CXCL10 is a pro-fibrotic factor, which participates in a crosstalk between hepatocytes, HSCs and immune cells. NK cells seem to play an important role in controlling HSC activity and fibrosis. CXCL10 blockade may constitute a possible therapeutic intervention for hepatic fibrosis.