Contribution of protein Z and protein Z-dependent protease inhibitor in generalized Shwartzman reaction.

OBJECTIVE: Sepsis, a leading cause of mortality in critically ill patients, is closely linked to the excessive activation of coagulation and inflammation. Protein Z, a cofactor for the protein Z-dependent protease inhibitor, enhances the inhibition of coagulation factor Xa, and protein Z-dependent protease inhibitor inhibits factor XIa in a protein Z-independent fashion. The functions of protein Z and protein Z-dependent protease inhibitor in the inflammatory and coagulant responses to septic illness have not been evaluated. DESIGN: For induction of generalized Shwartzman reaction, dorsal skinfold chamber-equipped mice were challenged twice with lipopolysaccharide (0.05 mg/kg on day -1 and 5 mg/kg body weight 24 hr later). Time-matched control animals received equal volumes of saline. SETTING: University research laboratory. SUBJECTS, INTERVENTIONS, AND MEASUREMENTS: Using intravital fluorescence microscopy in protein Z-dependent protease inhibitor deficient (ZPI) and protein Z deficient (PZ) mice, as well as their wild-type littermates (ZPI, PZ), kinetics of light/dye-induced thrombus formation and microhemodynamics were assessed in randomly chosen venules. Plasma concentrations of chemokine (C-X-C motif) ligand 1, interleukin-6, and interleukin-10 were measured. Liver and lung were harvested for quantitative analysis of leukocytic tissue infiltration and thrombus formation. MAIN RESULTS: After induction of generalized Shwartzman reaction, all mice showed significant impairment of microhemodynamics, including blood flow velocity, volumetric blood flow, and functional capillary density, as well as leukocytopenia and thrombocytopenia. Thrombus formation time was markedly prolonged after induction of generalized Shwartzman reaction in all mice, except of ZPI mice, which also had a significantly higher fraction of occluded vessels in liver sections. PZ mice developed the highest concentrations of interleukin-6 and interleukin-10 in response to generalized Shwartzman reaction and showed greater leukocytic tissue infiltration than their wild-type littermates. CONCLUSIONS: In this murine model of generalized Shwartzman reaction, protein Z-dependent protease inhibitor deficiency enhanced the thrombotic response to vascular injury, whereas protein Z deficiency increased inflammatory response.

Pivotal advance: alpha-galactosylceramide induces protection against lipopolysaccharide-induced shock.

alpha-galactosylceramide, a natural killer T cell ligand, and its synthetic homolog, KRN7000, consistently influence IFN-gamma and TNF-alpha release, both mediators of LPS-induced shock. To modify the course of endotoxin shock, we injected KRN7000 at different time points of experimental systemic Shwartzman reaction. Mice treated with KRN7000 survived when it was injected within 2 h before and after LPS challenge. Mice survival was associated with low levels of T helper 1 (Th1) cytokines, such as IFN-gamma and TNF-alpha. By contrast, protection from endotoxin shock was associated with an increase of T helper 2 (Th2) cytokines, like IL-4 and IL-10. A role of Th2 cytokines in counteracting LPS-induced shock was supported by experiments in which the protection against Shwartzman reaction by KRN7000 was abrogated by in vivo coadministration of anti-Th2 cytokines antibodies. In addition, cytofluorimetric analysis showed that surviving animals have higher percentages of NKT-IL-10-positive cells and lower percentages of NKT-IFN-gamma and macrophages/TNF-alpha-stained cells than nonprotected mice. Taken together, our data demonstrate that KRN7000 treatment given at times near LPS challenge is protective for endotoxin shock inhibiting IFN-gamma and TNF-alpha release. Moreover, KRN7000-mediated protection occurs through an increased production of IL-4 and IL-10, which are mainly secreted by NKT cells. Since IFN-gamma release by NKT requires a longer TCR stimulation than that required for Th2 cytokines production, we demonstrate that timing of KRN7000 in vivo exposure affect the pattern of cytokines expression protecting animals by endotoxin shock.

Depletion of plasma factor XIII prevents disseminated intravascular coagulation-induced organ damage.

The impact of clot stability affecting the vasculopathy and tissue necrosis in Shwartzman reaction was investigated using plasma Factor XIII A2-depleted rabbit (FXIII-DR). Plasma Factor XIIIA2 (FXIIIA2) was depleted by infusion of the mono-specific goat anti-rabbit FXIIIA2 IgG. Generalized Shwartzman reaction (GSR) was induced by priming and challenged by i.v. injection of LPS and local Shwartzman reaction (LSR) was primed by intradermal injection of LPS and challenged by i.v. injection of LPS. Histological examination of the GSR animals showed, extensive thrombi accumulation in renal tubules and bilateral cortical necrosis of kidney in 8 out of 10 rabbits but none in the FXIII-DR. Fibrinogen levels were elevated to 3 approximately 4 fold at 24 h and lowered at 48 h whereas a steady rise was seen in the FXIII-DR. FDP levels in GSR animals were significantly elevated at 24 h and further increased at 48 h but only slightly elevated in the FXIII-DR. Examination of the LSR tissues after 48 h showed an acute onset of progressive cutaneous vascular thrombosis, purpura, and secondary hemorrhagic necrosis whereas neither fibrin deposit nor necrosis of tissue were detected in FXIII-DR despite of an early edema formation. Fibrinogen levels were also increased two fold at 24 h but returned to basal levels at 48 h in control LSR animals but not affected at all in FXIII-DR. These results suggest that during the severe inflammatory conditions such as sepsis, the fibrinolytic system is functionally sufficient to dissipate the pathogenic accumulation of disseminated intravascular clots and exudated fibrin clots if those clots were prevented from getting crosslinked in plasma.

Inducción de la reacción de Shwartzman en el pulmón del conejo mediante el empleo de lipopolisacárido de Pasteurella haemolytica / Induction of the Shwartzman reaction in the rabbit lung employing Pasteurella haemolytica lipopolysacharide

Se indujo la reacción de Shwartzman (RS) en el pulmón de conejo empleando lipopolisacárido (LPS) de Pasteurella haemolytica con el fin de comparar las lesiones provocadas con las que se presentan naturalmente en la pasteurelosis neumónica (PN). Para inducirla, primero se administró una dosis de LPS de Pasteurella haemolytica (50 mg) por vía endotraqueal (inoculación preparatoria) seguida 24 h más tarde por otra dosis del mismo LPS (100 mg) por vía endovenosa (inoculación desencadenante) (Grupo 5). Doce horas después, los animales fueron sacrificados. El pulmón derecho se destinó a estudios de histopatología, mientras que el izquierdo se empleó para realizar lavados bronquioalveolares (LBA). Para comparar se incluyeron grupos que recibieron solamente el LPS de P. haemolytica por vía endotraqueal (Grupo 3) o endovenosa (Grupo 4) (la inoculación faltante correspondió a solució salina fisiológica [SSF]), así como otro grupo al que se le administró LPS de Escherichia coli tal y como se describió al principio para inducir la RS (Grupo 2), y otro más que recibió SSF de la misma manera (Grupo 1). Las lesiones más notorias se presentaron en los animales que recibieron LPS de P. haemolytica por vía endotraqueal y en los que se les provocó la RS con el mismo LPS. Sólo los polimorfonucleares (PMN), monocitos y linfocitos recuperados por LBA resultaron diferentes entre los grupos, particularmente en los animales que recibieron el LPS de P. Haemolytica por vía endotraqueal y a los que se les indujo la RS con el mismo LPS. Se concluye que el LPS de P. Haemolytica es capaz de provocar una reacción flogística en el pulmón tanto por vía endotraqueal como endovenosa, aunque la respuesta es mucho más intensa en la primera, además, la RS no provoca una respuesta inflamatoria más intensa que la sola inoculación endotraqueal del LPS. Finalmente, se puede afirmar también que el conejo es un buen modelo para evaluar la respuesta inflamatoria pulmonar provocada por el LPS de P. haemolytica

Local skin reactivity after induction of Shwartzman reaction in rabbits.

A local Shwartzman response was elicited in rabbits by an intradermal injection of the Salmonella typhosa endotoxin lipopolysaccharide (LPS) followed 24 hours later by an intravenous challenge injection with zymosan. After the intravenous challenge, necrotizing vasculitis developed in the prepared skin sites which was characterized by microthrombi, accumulation of neutrophil granulocytes, fibrin deposition and extravasation of red blood cells. Evans' blue extravasation into the altered tissue was significantly reduced, and histologically, the intensity of the Shwartzman reaction in the skin was reduced by pretreatment with thalidomide and dexamethasone. The mechanism of reduction of an LPS-induced local Shwartzman reaction by thalidomide is discussed.

Lesions resulting from attempted Shwartzman reaction in turkey poults inoculated with Pasteurella multocida lipopolysaccharide.

Five-week-old turkey poults were given two consecutive intravenous injections (24 hours apart) of highly purified Pasteurella multocida lipopolysaccharide (LPS) in an effort to induce a generalized Shwartzman reaction. There were no gross lesions, and microscopic lesions were limited to focal hepatic necrosis with heterophil infiltration. Hepatic lesions did not differ qualitatively from lesions in turkeys given a single dose of lipopolysaccharide. Margination of heterophils in the pulmonary vasculature was observed in turkeys 4 hours after a single injection of LPS, but it was not present in turkeys given the consecutive injections of LPS. To induce a dermal Shwartzman reaction, turkeys were given intradermal injections of LPS followed by an intravenous injection of LPS 24 hours later. Although no grossly visible hemorrhagic dermal necrosis occurred, microscopic lesions, including heterophil infiltration, vasculitis, thrombosis, and necrosis, were present. Thrombosis and vasculitis were observed only in turkeys given the intravenous and intradermal LPS, whereas the other inflammatory changes were observed in turkeys given the intradermal injection of LPS and intravenous water. Prominent lymphocytic perivascular cuffing at the site of dermal injection was present in all turkeys given intradermal LPS.

Biological activities--lethality, Shwartzman reaction and pyrogenicity--of Salmonella typhimurium porins.

Porins isolated from Salmonella typhimurium and found to contain less than 0.1% w/w of LPS, were found to be lethal at a dose of 100 ng to both LPS-responder (BALB/cByJ) and non-responder (C3H/HeJ) mice sensitized with D-galactosamine. This lethal action could be prevented by anti-TNF-alpha serum given intravenously 10 min before the porin injection but not by polymyxin-B mixed with the porins in a ratio of approximately 300 moles polymyxin-B per mole of porin. The porin preparation was also pyrogenic to rabbits at a dose of 1 microgram/kg and elicited a local Shwartzman reaction when used as the sensitizing and eliciting agent; these reactions were also present when the porins were mixed with polymyxin-B.

Biological activities of lipopolysaccharide isolated from Vibrio cholerae O139, a new epidemic strain for recent cholera in Indian subcontinent.

Biological activities of lipopolysaccharide (LPS) isolated from Vibrio cholerae O139, a new causative agent for recent cholera epidemic in Indian subcontinent, were investigated in comparison with those of LPS from O1 V. cholerae. V. cholerae O139 LPS exerted mitogenic activity, lethal toxicity and Shwartzman reaction to the same extent as those observed for O1 V. cholerae LPS, although these activities except for lethal toxicity were obviously lower than those of Salmonella typhimurium LT-2 LPS used as a reference. It was, therefore, suggested that O139 LPS does not contribute to the high infective and pathogenic potentials of the V. cholerae O139 strain as in the case of O1 V. cholerae.

Chemical and biological properties of endotoxin from Leptospira interrogans serovars canicola and icterohaemorrhagiae.

1. Endotoxin-like activity was extracted with phenol-chloroform-petroleum either (PCP) from Leptospira interrogans serovars icterohaemorrhagiae and canicola. Chemical analysis of leptospiral cells obtained from the PCP extract indicated the following distribution of lipopolysaccharide (LPS), protein and polysaccharide in mg/ml: 3.0, 4.5 and 1.0 for icterohaemorrhagiae and 3.3, 5.6 and 1.5 for canicola. 2. The preparations presented several biological activities: positive Limulus test (1.0 pg/ml) for icterohaemorrhagiae and canicola PCP extract and 0.5 pg/ml for E. coli O111:B4 LPS, lethality for chicken embryos (LD50 45, 25 and 1.0) for icterohaemorrhagiae, canicola and E. coli O111:B4 LPS, pyrogenicity in rabbits with an average increase in rectal temperature of 0.6 degrees C, 0.9 degrees C and 2.2 degrees C for canicola, icterohaemorrhagiae and E. coli O111:B4 LPS, reacted with complement inhibiting the lysis of sheep red blood cells, 62%, 75% and 90% for 2.0 micrograms/ml of icterohaemorrhagiae, canicola PCP extract and E. coli O111:B4 LPS. The PCP extract showed no cytotoxicity on chicken embryo fibroblasts and epithelial cells. 3. These results demonstrate that Leptospira endotoxin activity is similar to E. coli O111:B4 lipopolysaccharide.

Increased susceptibility of aged rats to haemorrhage and intravascular hypercoagulation following endotoxin administered in a generalized Shwartzman regime.

Ageing rats are known to have an increased incidence of myocardial fibrosis and dyspnoea caused by pulmonary intravascular coagulation. In order to determine whether endotoxin can be responsible for such responses in ageing rats we have exposed rats of differing ages (2 months, 16 months and 24 months) to single or repeated (two doses 24 h apart; generalized Shwartzman regime) intravenous doses of endotoxin (E. coli 0111 B4). Only the 2-year-old rats reacted adversely. Two doses of endotoxin produced death, with focal myocardial necrosis, haemorrhage and pulmonary and hepatic intravascular coagulation. The increased susceptibility of aged rats to the toxic effects of endotoxin explains some of the changes found in the tissues of old rats. The sporadic nature of both cardiac failure and dyspnoea as a cause of morbidity and mortality in ageing rats may be related to the need for two endotoxin episodes in a period of 24 h to provoke a generalized Shwartzman reaction, an occurrence likely to be relatively uncommon under natural conditions.

Prevention of human TNF-induced cutaneous Shwartzmann reaction and acute mortality in mice treated with anti-human TNF monoclonal antibodies.

We studied monoclonal antibodies (MoAbs) directed against human tumour necrosis factor (TNF) for their capacity to prevent toxic or lethal effects of TNF. Two experimental models involving recombinant human TNF (rhTNF) in mice were used: the Shwartzmann reaction, and the lethality after D-galactosamine sensitization. Two MoAbs were found to be protective in both models. These MoAbs prevented mortality when given 6 h, 4 h, or 15 min before rhTNF injection but were not effective if given after TNF. In addition, our results point out that in vitro binding and even neutralizing capacities of anti-hTNF MoAbs do not necessarily reflect their protective efficacy in vivo. Therefore, the models studied here might be useful to evaluate anti-h TNF MoAbs before clinical use.

Cholera and pertussis exotoxins protect mice against the lethal Schwartzman reaction and antagonize the effects of lipopolysaccharide on second messenger systems.

Pertussis toxin, and also cholera toxin are capable of inhibiting the effects of LPS in the elicitation of the generalized Schwartzman reaction. This is a potentially lethal generalized thrombo-haemorrhagic hypersensitivity and inflammatory-type response that occurs after two consecutive injections of LPS. The two exotoxins furnish significant protection against the lethal outcome of this reaction. It is known that the acute haematological and haemodynamic changes are accompanied by alterations in the levels of various endogenous mediators: glucocorticoid hormones, prostaglandins, arachidonic acid metabolites, cytokines and proteases. In vitro effects of LPS on murine leukocyte cell lines can be antagonized by pertussis toxin, implicating a Gi-like regulatory protein in the mediation of these effects. Experiments designed to study the involvement of particular second messenger systems (cAMP and phosphatidylinositol) used by LPS in vivo, revealed that the protective effects conferred by these exotoxins are associated with the antagonization of alterations caused by LPS. No correlation was found between the levels of IL-6 and the mortality rate in this experimental mouse model. The results indicate that G proteins play a role in the generation of the Schwartzman reaction and open a new approach for pharmacological intervention in endotoxemia and in clinical settings with Gram-negative sepsis.

Shwartzman reaction in the brain induced by fractions of Fusobacterium necrophorum and Escherichia coli lipopolysaccharide in rabbits.

Multifocal fibrin thrombosis and suppurative meningitis in the central nervous system was induced by intracerebral inoculation with a cytoplasmic or supernatant fraction of Fusobacterium necrophorum followed by intravenous inoculation with Escherichia coli lipopolysaccharide endotoxin in rabbits. Formation of fibrin thrombosis was reduced by heparin administration. Single intracerebral inoculation with the cytoplasmic or supernatant fraction caused suppurative meningitis. Nervous lesions were often associated with fibrinoid degeneration of the blood vessels. Bacterial lipopolysaccharide endotoxin did not induce apparent meningitis or fibrin thrombosis. The formation of fibrin thrombosis in the central nervous system might be attributable to the Shwartzman reaction.

Importance of fatty acid substituents of chemically synthesized lipid A-subunit analogs in the expression of immunopharmacological activity.

The immunopharmacological activities of chemically synthesized lipid A-subunit analogs, 4-O-phosphono-D-glucosamine derivatives carrying different N- and 3-O-linked acyl groups, were investigated. None of the synthetic compounds tested exhibited any detectable pyrogenicity at a dose of 10 micrograms/kg. Weaker lethal toxicity in galactosamine-sensitized mice was detected at 1 microgram per mouse for all the synthetic compounds except GLA-58. Among (RS) stereoisomers of 4-O-phosphono-D-glucosamine derivatives carrying a 3-O-tetradecanoyl (C14) group with different N-linked acyloxyacyl groups, i.e., 3-dodecanoyloxytetradecanoyl [C14-O-(C12)], 3-tetradecanoyloxytetradecanoyl [C14-O-(C14)], and 3-hexadecanoyloxytetradecanoyl [C14-O-(C16)] groups (termed GLA-57, GLA-27, and GLA-58, respectively), GLA-27 exhibited significant colony-stimulating factor-inducing and tumor necrosis factor-inducing activities, mitogenicity, polyclonal B-cell activation activity, macrophage activation, and adjuvanticity. The activities of GLA-57, which had an N-linked C14-O-(C12) group, were equivalent to or somewhat weaker than those of GLA-27 with a C14-O-(C14) group. Significant immunopharmacological activities were not observed for GLA-58, carrying a C14-O-(C16) group bound to the amino group. GLA-59, carrying 3-O-linked 3-hydroxytetradecanoyl (C14OH) and N-linked C14-O-(C14) groups, showed much higher activities than GLA-27, GLA-60, a compound which possesses the same fatty acid substituents as GLA-59 but with reversed binding sites, showed the strongest B-cell activation and adjuvant activities among the synthetic compounds. Among stereoisomers of GLA-59 and GLA-60 composed of fatty acid substituents with the (RR) and (SS) configuration, compounds with the (RR) configuration elicited stronger activities than the (SS) stereoisomers. The importance of fatty acid substituents, including stereospecificity for the expression of immunopharmacological activities of 4-O-phosphono-D-glucosamine derivatives, was demonstrated.

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

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 the zona 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.

Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A.

A synthetic lipid A (preparation 516), containing seven acyl groups and representing one component of natural free lipid A of Salmonella minnesota R595, has been investigated for biological activity in a number of endotoxin test systems. It was found that the synthetic preparation was, in typical in vivo endotoxin tests (lethality, pyrogenicity, Shwartzman reactivity) as well as in its antigenicity and macrophage activation capacity, significantly less active than natural Salmonella lipid A. However, in other in vitro assay systems (B-cell mitogenicity, complement activation, Limulus amoebocyte lysate gelation) it expressed similar activity as Salmonella lipid A.

Structure-activity relationship of lipid A: comparison of biological activities of natural and synthetic lipid A's with different fatty acid compositions.

To investigate the structure-activity relationships, various biological activities, including pyrogenicity, lethal toxicity, elicitation of Shwartzman reaction, mitogenicity and tumor necrosis factor (TNF)-inducing activity, were compared among natural and synthetic lipid A's differing in fatty acid composition. In all these tests, natural lipid A's from Escherichia coli and Salmonella minnesota and synthetic LA-15-PP, which carries 3-hydroxy- and 3-acyloxy-tetradecanoyl groups at the 2, 3 and 2', 3' positions, respectively, showed the strongest activities among the tested lipid A's. In contrast, LA-16-PP, in which the amide-bound 3-hydroxytetradecanoic acid at position 2 of LA-15-PP is replaced by 3-hexadecanoyloxytetradecanoic acid, exhibited lower activity than LA-15-PP and natural lipid A's. Although LA-16-PP has been assumed to have a typical Salmonella lipid A structure (and, in fact, it has a structure corresponding to one of the components of Salmonella lipid A), the activity of this synthetic compound was not comparable to that of natural Salmonella lipid A. LA-17-PP, in which tetradecanoic acid is the sole fatty acid component, exhibited relatively strong mitogenicity and TNF-inducing activity, but very low pyrogenicity. The activities of LA-18-PP, which has ester-bound tetradecanoic acid and amide-bound 3-hydroxytetradecanoic acid, were lower than those of LA-17-PP. The results indicate that the differences in fatty acid composition of lipid A's have important influences on the biological activities studied.

Synthetic lipid A with endotoxic and related biological activities comparable to those of a natural lipid A from an Escherichia coli re-mutant.

A synthetic compound (506), beta (1-6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, exhibited full endotoxic activities identical to or sometimes stronger than those of a reference lipid A from an Escherichia coli Re-mutant (strain F515). Endotoxic activities tested include pyrogenicity and leukopenia-inducing activity in rabbits, body weight-decreasing toxicity in normal mice, lethal toxicity in galactosamine-sensitized mice and chicken embryos, and the preparation and provocation of the local Shwartzman reaction in rabbits. Compound 406, a synthetic counterpart of a biosynthetic precursor of lipid A molecule, showed by contrast only weak activities in all of the above assay systems except for the lethality in galactosamine-loaded mice. This finding strongly suggests that the presence of acyloxyacyl groups at the C-2' and C-3' positions of the disaccharide backbone is one of the most important determinant structures of the lipid A molecule for exhibition of strong biological activities characteristic of lipopolysaccharide and its lipid A moiety. The activities of the corresponding 4'-monophosphate (compound 504) and 1-monophosphate (505) analogs were considerably less than those of the parent molecule 506 and the reference F515 lipid A. Regarding other biological activities, not only compound 506 but also compounds 504, 505, and 406 showed definite activities, sometimes comparable to those of F515 lipid A and other reference natural products. These are the activation of Tachypleus tridentatus amoebocyte clotting enzyme cascade and human complement via the classical pathway, mitogenic and polyclonal B-cell activation of murine splenocytes, stimulation of peritoneal macrophages in a guinea pig, enhancement of migration of human blood polymorphonuclear leukocytes, and induction of a serum factor that is cytostatic and cytocidal to L-929 cells in Mycobacterium bovis BCG-primed mice. Relative potencies of test synthetic compounds depended on the assay systems and varied from one system to another. Dephospho-compound 503 lacked most of the biological activities that were definitely observed with phosphorylated compounds, probably because of its insolubility. This study demonstrates the successful chemical synthesis of an E. coli-type lipid A.