Induction of Src-suppressed C kinase substrate (SSeCKS) in vascular endothelial cells by bacterial lipopolysaccharide.

We isolated cDNA of the mouse homologue of the src-suppressed C kinase substrate (SSeCKS) and analyzed the effects of lipopolysaccharide (LPS) injection on the tissue expression pattern of this protein. Northern blotting analysis showed that SSeCKS mRNA was expressed abundantly in the testis but at undetectable levels in other tissues of untreated control mice. Intraperitoneal administration of LPS strongly induced SSeCKS mRNA expression in the lung, heart, liver, spleen, kidney, lymph node, adrenal gland, and pituitary gland, as well as in the brain. In lung and spleen, the SSeCKS mRNA levels increased almost 10-fold at 1 hr after LPS injection and persisted at high levels until 4 hr. Both in situ hybridization and immunohistochemical studies revealed that LPS administration conspicuously elevated expression of SSeCKS mRNA and protein in vascular endothelial cells of several organs. Ectopic expression of SSeCKS caused loss of cytoplasmic F-actin fibers in the mouse endothelial cell line LEII. These results indicate that SSeCKS is one of the major LPS-responsive proteins and may participate in alteration of cytoskeletal architecture in endothelial cells during inflammation.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. I. Enhanced production of interferon and appearance of cytotoxin stimulated by capsular polysaccharide of Klebsiella pneumoniae or bacterial lipopolysaccharide.

Interferon production stimulated by the active substance (neutral fraction) of the capsular polysaccharide of Klebsiella pneumoniae (neutral CPS-K) in BCG-infected mice was compared with that by bacterial lipopolysaccharide (LPS). Prior infection with BCG increased the responsiveness of mice to the lethal effect of neutral CPS-K as well as to that of LPS. Associated with this, BCG-infected mice showed a markedly enhanced ability to produce interferon after stimulation not only by LPS but also by neutral CPS-K. In addition, a cytotoxic factor (cytotoxin) was found to be released in the serum of BCG-infected mice after injection of these inducers. The kinetics of production of interferon and cytotoxin stimulated by neutral CPS-K were very similar to those stimulated by LPS. The time pattern of cytotoxin production was not in parallel with that of interferon production. Interferon reached a peak 2 hr and cytotoxin 3 hr after injection with these inducers. Interferon and cytotoxin produced by neutral CPS-K showed essentially the same stabilities to heating at 56 C and to treatment at pH 2 respectively as those produced by LPS. Interferon was inactivated by heating at 56 C more rapidly than cytotoxin. Cytotoxin was inactivated by treatment at pH 2 for 24 hr, whereas interferon activity was well preserved after this treatment. These results suggest that both activities are the result of different substances.     

Localization of intravenously injected lipopolysaccharide (LPS) in the spleen of the mouse. An immunoperoxidase and histochemical study

In earlier studies we investigated the in vivo effects of lipopolysaccharide (LPS) on lymphoid and non-lymphoid cells in the mouse spleen. In order to find out whether LPS localizes in and/or on cells that are affected by this compound, the aim of the present study was to investigate the localization of intravenously injected LPS in the mouse spleen using an immunoperoxidase technique. At different time points after injection, the localization of LPS is demonstrated and LPS-containing cells are characterized. Most of the injected LPS has been taken up by marginal zone macrophages at 2 h after its administration whereas macrophages in the red pulp and at the periphery of the white pulp (marginal metallophils) have ingested less LPS. In the periarteriolar lymphocyte sheath, LPS is concentrated in a large number of acid phosphatase-negative, Ia-positive, large branched cells which were suggested to represent interdigitating cells. Moreover an extracellular dendritic localization pattern of LPS is demonstrated in the corona and central parts of the follicles at different time intervals after its injection. The significance of the localization pattern of LPS in the mouse spleen is discussed.     

FURTHER STUDIES ON MOBILIZATION OF CFUs

Mobilization of CFUs from haemopoietic tissues into circulation was studied after injection of different bacterial lipopolysaccharides (LPS), zymosan, phytohaemagglutinin (PHA), concanavalin A (Con A), trypsin and di-isopropyl-fluorophosphate-inhibited trypsin. All bacterial LPS used gave an increase of CFUs in the peripheral blood at 1 h after i.v. injection. Some variation in activity could not be excluded. As with Salmonella typhosa LPS, zymosan gave an increase in circulating CFUs during the first few hr and a second peak a few days later. After injection of zymosan as well as S. typhosa LPS the second peak in the blood was accompanied by a large increase in CFUs numbers in the spleen. PHA gave an immediate mobilization of CFUs, but the mobilization after injection of Con A during the first few hr occurred more slowly. After injection of S. typhosa LPS, zymosan and PHA the blood C3 level was found to be depressed considerably. This might indicate that the complement system is involved in the early mobilization of CFUs. Dexamethasone, a synthetic hormone which has been reported to give sequestration of several cell types in the bone marrow, did not inhibit the early and late mobilization of CFUs which normally occurs after injection of S. typhosa LPS.     

Endotoxin inactivating activity of rat serum

The ability of rat serum to inactivate endotoxin (LPS) was assessed with the aid of the limulus amebocyte lysate assay. Following the addition of various amounts of endotoxin to normal serum the mixture was incubated for 1 hr at 37 degrees C and the residual endotoxin activity determined. One milliliter of rat serum inactivated between 5 and 10 micrograms Escherichia coli LPS per hour. Heating serum for 45 min at 56 degrees C resulted in loss of 80-90% of the LPS inhibitor (LPSI) activity. Serum from cobra venom factor (CVF)-treated rats inactivated between 0.5 and 2.5 micrograms LPS/ml serum. Serum from tolerant rats, even after heating for 45 min at 56 degrees C, inactivates between 10 and 15 micrograms LPS/ml serum/hr; decomplemented tolerant rat serum neutralizes between 5 and 10 micrograms LPS/ml serum/hr. Clearly, the tolerant rat has large quantities of LPSI activity, which does not appear to be complement. The inhibitor found in tolerant rat serum is not species specific since it inactivates Salmonella minnesota and Salmonella typhimurium endotoxins to the same degree and in the same amount as E. coli endotoxin, the agent used to induce tolerance. Both heating serum (56 degrees C) and lead acetate reduce LPSI activity.     

Effects of bacterial lipopolysaccharide and calmodulin on Ca2(+)-ATPase and calcium in human natural killer cells, studied by a combined technique of immunoelectron microscopy and ultracytochemistry

Our previous studies indicate that bacterial lipopolysaccharide (LPS) enhances natural killer (NK) cell-mediated cytotoxicity and increases intracellular calcium (Ca2+) in hepatocytes. Calmodulin (CAM) regulates Ca2(+)-ATPase activity, intracellular Ca2+, and is also implicated in NK cell-mediated cytolysis. In the present work, the effects of LPS and CAM on Ca2(+)-ATPase and intracellular Ca2+ in human NK cells were studied by a combined technique of immunogold electron microscopy and ultracytochemistry. Peripheral blood mononuclear cells were treated with 100 micrograms/ml E. coli (0111:B4) LPS and/or 5 micrograms/ml CAM in RPMI 1640 medium at 37 degrees C for 1 or 4 hr. NK cells labeled with monoclonal anti-Leu-11a (CD16) antibody and colloidal gold-conjugated anti-mouse IgG were processed for cytochemical localization of Ca2(+)-ATPase and Ca2+. Ca2(+)-ATPase was localized in the plasma membrane of NK cells, and its activity was suppressed by LPS but was enhanced by CAM. However, no apparent changes in the enzyme reaction were observed when cells were exposed to CAM concomitantly with LPS or stimulated with LPS before CAM. Apparent reduction of the enzyme reaction was observed when LPS stimulation was preceded by CAM. Ca2(+)-ATPase reaction in mitochondria was observed only in NK cells exposed to CAM. Computer image analysis showed no changes in the intracellular Ca2+ in NK cells treated with LPS for 1 hr, whereas a significant increase in Ca2+ was found in cells exposed to LPS for 4 hr. The intracellular Ca2+ significantly decreased in NK cells treated with CAM or with a combination of LPS and CAM as compared to that of controls (p less than 0.05). The results indicate that CAM is capable of blocking or reversing the inhibitory effect of LPS on Ca2(+)-ATPase, and suggest that in human NK cells the plasma membrane-associated Ca2(+)-ATPase is responsible for extrusion of intracellular Ca2+.     

In vivo response of neutrophils and epithelial cells to lipopolysaccharide injected into the monkey ileum

Since few previous studies have investigated the in vivo response of intestinal mucosa to the luminally administered lipopolysaccharide (LPS), we examined the cellular localization of exogenously applied LPS in the intestinal mucosa and the expression of Toll-like receptor (TLR) and IL-1 receptor-associated kinase (IRAK) in the epithelial cells of monkey ileum. FITC-labeled LPS was injected into the lumen of monkey ileum. Thirty minutes after the LPS injection, the ileal tissue was fixed and localization of FITC fluorescence in the ileal mucosa was examined. We applied Factor C immunohistochemistry to demonstrate the bioactivity of LPS taken up by the mucosal tissue. The expression of TLR4 and IRAK-1 in the epithelial cells was also examined by immunohistochemistry. FITC fluorescence was detected in the cells migrated into the epithelium and those in the lamina propria. The FITC-labeling cells were completely overlapped with the Factor C immunoreactive cells. These FITC-labeling/Factor C-positive cells were identified as neutrophils by the immunoelectron microscopic analysis. TLR4 and IRAK-1 were expressed at the apical membrane of the epithelial cells in the ileum of both control and FITC-LPS injected animals. These results suggest that intraluminal injection of LPS stimulates the transmigration of neutrophils into the epithelium and these neutrophils may uptake luminally applied LPS and possibly inactivate the enterotoxin. Expression of TLR4 and IRAK-1 in the epithelial cells suggests that epithelial cells may react to LPS and produce chemoattractant mediator to induce the neutrophil chemotaxis.     

c-FOS protein expression in cells of the variuos hypothalamic structures after electric pain stimulation and injections of antigens

Hypothalamic structures become activated after stimul. Alteration of c-Fos-positive cells quantity in different hypothalamic structures after electric pain stimulation (EPS), intravenous (iv) injection of antigens (lioppolysaccharide (LPS) and bovine serum albumir (BSA)) was detected with immunohistochemical method. EPS and iv injection of antigens (LPS and BSA) result in c-Fos-positive cells quantity increase in all observed hypothalamic structures. The highest activation level was in AHN and PH after EPS and in AHN, PVH, LHA-28, and PH after iv LPS injection. Comparative analysis of results showed, that c-Fos-positive cells quantity increase after EPS in AHN, PVH, LHA and PH was more significant than after iv injection of antigens (LPS and BSA). LPS injection results in more pronounced cell activation in AHN, PVH, LHA-28 and DMH (according to quantity of c-Fos-positive cells), than BSA injection.     

Regulation of brain cell environment on neuronal protection: role of TNFalpha in glia cells

Bacterial endotoxin lipopolysaccharide (LPS) treatment of neuron-rich cells and glia-rich cells exhibited significant cell damage 12 hr after incubation, although no severe or significant cell damage induced by LPS appeared in neuron-glia co-cultured cells. Moreover, severe and significant time-dependent cell damage was induced by a larger dose treatment (10 mM) of glutamate (Glu), and this damage was seen in neuron-rich cells, neuron-glia co-cultured cells, and glia-rich cells. Examining extracellular tumor necrosis factor alpha (TNFalpha) induced by either LPS or Glu treatment, the levels of extracellular TNFalpha induced by LPS were significantly higher than those induced by Glu. These significant increases of TNFalpha were measured within 2 hr after LPS treatment in neuron-glia co-cultured cells and glia-rich cells, although no significant changes were detected in the neuron-rich cells. With Glu treatment, a significant increase in TNFalpha levels was detected after 6 hr of Glu treatment only in glia-rich cells. Our results indicate that cerebral TNFalpha is mainly produced in glia cells and that its production is dependently regulated by each stimulant. In addition, the production of TNFalpha is not directly related to the trigger of cell injury.     

Further studies on mobilization of CFUs.

Mobilization of CFUs from haemopoietic tissues into circulation was studied after injection of different bacterial lipopolysaccharides (LPS), zymosan, phytohaemagglutinin (PHA), concanavalin A (Con A), trypsin and di-isopropyl-fluorophosphate-inhibited trypsin. All bacterial LPS used gave an increase of CFUs in the peripheral blood at 1 h after i.v. injection. Some variation in activity could not be excluded. As with Salmonella typhosa LPS, zymosan gave an increase in circulating CFUs during the first few hr and a second peak a few days later. After injection of zymosan as well as S. typhosa LPS the second peak in the blood was accompanied by a large increase in CFUs numbers in the spleen. PHA gave an immediate mobilization of CFUs, but the mobilization after injection of Con A during the first few hr occurred more slowly. After injection of S. typhosa LPS, zymosan and PHA the blood C3 level was found to be depressed considerably. This might indicate that the complement system is involved in the early mobilization of CFUs. Dexamethasone, a synthetic hormone which has been reported to give sequestration of several cell types in the bone marrow, did not inhibit the early and late mobilization of CFUs which normally occurs after injection of S. typhosa LPS.     

Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo

A highly specific radioreceptor assay for cachectin/tumor necrosis factor (TNF) was utilized to measure the time course of lipopolysaccharide (LPS)-induced hormone production in rabbits. Cachectin/TNF bioactivity was monitored in the same serum samples by measuring lipoprotein lipase (LPL) suppression in 3T3-L1 cells. Cachectin/TNF is produced in large quantities by LPS-treated rabbits without priming by bacillus Calmette Guérin, C. parvum, or other agents. Nanomolar concentrations of the hormone are achieved, with peak levels occurring at 2 hr postinjection; the hormone is rapidly cleared thereafter. In separate studies, mice were used to assess the distribution and metabolic fate of cachectin/TNF. Radioiodinated hormone is cleared from the plasma with a half-life of 6 to 7 min. Studies of the tissue distribution of label after injection demonstrate that liver, kidneys, skin, and gastrointestinal tract take up most of the hormone. Electrophoretic analysis of tissues recovered from injected animals suggests that the hormone is very rapidly degraded after binding.     

Localization of brain-type fatty acid-binding protein in Kupffer cells of mice and its transient decrease in response to lipopolysaccharide

Brain-type fatty acid-binding protein (B-FABP) was localized in Kupffer cells of liver of postnatal day 10 (P10) and older mice in immunolight and electron microscopy as well as by in situ hybridization histochemistry. The immunoreaction products were localized in the cytoplasmic matrix but not within the nucleus. After peritoneal injection of lipopolysaccharide (LPS), the immunoreaction for B-FABP decreased markedly in Kupffer cells at 1 h postinjection and thereafter gradually recovered to the preinjection level by 24 h postinjection, although no decrease in the mRNA expression was detected in Northern blotting throughout the course after the injection. The specific localization of B-FABP, but not the other FABPs, in Kupffer cells, and its rapid decrease after LPS injection suggest the intimate involvement of B-FABP in Kupffer cells in the inflammatory reaction, probably through mediation of n-3 polyunsaturated fatty acids, which are strong binders of B-FABP.     

Histological analysis of mammary gland remodeling caused by lipopolysaccharide in lactating mice

The mammary alveolus is a highly specialized structure that secretes milk for suckling infants during lactation. The secreting alveolus consists in alveolar epithelial cells (AECs) and myoepithelial cells and is surrounded by microvascular endothelial cells, adipocytes and several immune cell types such as macrophages and neutrophils. During normal lactation, these cells play distinct roles needed to maintain the secretory ability of the mammary alveolus. However, inflammation resulting from pathogenic bacterial infections causes structural and functional regression of the secreting alveolus in the lactating mammary gland. We initiated artificial inflammation in the mammary glands of lactating mice by injecting lipopolysaccharide (LPS), as a mammary inflammation model and investigated, by immunohistochemical analysis, the early response of the cells constituting and surrounding the alveolus. Some AECs sloughed away from the alveolar epithelial layer and showed progression of apoptosis detected by immunostaining of cleaved caspase-3 after LPS injection. Adipocytes exhibited transient shrinkage and re-accumulation of lipid droplets, although the numbers of adipocytes did not demonstrate a significant difference. Activation of F4/80-positive cells around the mammary alveolus was observed 3 h after LPS injection. However, the recruitment of CD11b-positive cells into the alveolar lumen was not observed until 12 h after LPS injection. Myoepithelial cells were contracted after LPS injection. LPS injection around the alveolus did not induce any detectable structural changes in capillaries surrounding the alveolus. Thus, cell-specific behavior and tissue remodeling of the alveolus occur after LPS injection in a time-dependent manner.     

Down-regulation of Fc receptor expression in guinea pig peritoneal exudate macrophages by muramyl dipeptide or lipopolysaccharide

Expression of Fc receptors on the plasma membrane of guinea pig peritoneal exudate macrophages (PEM) was suppressed to almost one-half of that of the controls by long-term exposure to lipopolysaccharide (LPS) or muramyl dipeptide (MDP) in culture. The effect of the reagents was dose and time dependent, and as little as 0.5 ng/ml LPS or 5 ng/ml MDP was effective for the suppression. The expression of the Fc receptors decreased to 60 to 70% of the control level at 48 hr and to 45 to 50% at 72 hr after incubation of the cells in the presence of LPS or MDP. A Scatchard plot of the binding of 125I-soluble immune complexes (I.C.) to the cells revealed that the decrease in the binding of 125I-I.C. is due to a reduction in the number of Fc receptors on the cell membrane and not to a decreased affinity of the receptors. The membrane protein was radio-labeled with 125I, and the Fc receptors were purified by being bound to insoluble I.C. The specific binding of the 125I-labeled Fc receptors, from the LPS-treated macrophages, to the insoluble I.C. was almost one-half of that from the untreated control cells. SDS-PAGE analysis of the purified 125I-labeled Fc receptors revealed that the major peak of the m.w. 44,000 molecule in the LPS-treated cells was almost one-half of that of the control. Contrary to the effect of LPS or MDP, 72-hr incubation of macrophages with MIF-rich supernatant, cultured from lymph node cells, enhanced the expression of Fc receptors. Macrophages were treated with I.C. for 4 hr at 37 degrees C to remove the Fc receptors from the surface membrane. The reappearance of the receptors on the plasma membrane of the cells was significantly suppressed by LPS and MDP. The effect of LPS on the binding of five murine monoclonal antibodies (Ab) raised against PEM to the macrophage membrane and also that of 125I-wheat germ agglutinin (WGA) or 125I-insulin was studied. The monoclonal Ab were selected for their activity to induce superoxide anion generation in the macrophages, as do I.C., although the binding sites for the monoclonal Ab were not related to Fc receptors. The bindings of the five monoclonal Ab were not affected by exposure of the cells to LPS or MDP. Macrophages treated with the reagents bound as much 125I-insulin or WGA as did the untreated control cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

THE MIGRATION OF LYMPHOCYTES ACROSS SPECIALIZED VASCULAR ENDOTHELIUM

Lymphocytes were exposed in vitro to either trypsin or neuraminidase. The ability of the treated cells to migrate into tissues was measured (a) by i.v. injection into intact recipients and (b) by vascular perfusion through an isolated lymph-node preparation. The localization of trypsinized cells in the lymph-nodes of recipients was deficient when compared to untreated lymphocytes and there was a surplus of trypsinized cells in the blood. Trypsinized cells migrated into the isolated nodes in reduced numbers. By contrast, neuraminidase treated lymphocytes were markedly deficient in the blood of recipients early after injection; their localization in the spleen and lymph-nodes was also deficient but they were in surplus in the liver. Moreover they migrated into the isolated nodes in slightly increased numbers. By 24 hr after injection the perturbed localization pattern produced by either enzyme was partly restored to normal. In conclusion, trypsin interfered with the capacity of lymphocytes to migrate into lymph-nodes but neuraminidase did not; the latter promoted the hepatic sequestration of cells and the reduced localization in the blood and tissues was a consequence of this. The hypothesis that lymphocytes adhere to specialized endothelia in lymph-nodes because of specific glycoside sequences on their surface lacks experimental support.