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.

Exacerbation of vascular endothelial injury in the generalized Shwartzman reaction by the administration of anti-E-selectin antibody.

Previously, we reported that the consecutive administration of lipopolysaccharide (LPS) into LPS-sensitized mice for the generalized Shwartzman reaction (GSR) induced systemic injury of vascular endothelial cells. The aim of this study was to investigate the participation of vascular adhesion molecules in the vascular endothelial injury of GSR. The administration of anti-E-selectin antibody in GSR-induced mice resulted in massive apoptosis of vascular endothelial cells and congestion in blood vessels. Further, marked hemorrhage was found in the pulmonary alveoli of those mice. GSR, especially lung injury, was definitely exacerbated by the administration of anti-E-selectin antibody. On the other hand, the administration of anti-VCAM-1 antibody did not induce such injury of vascular endothelial cells. The possible role of E-selectin in the exacerbation of vascular endothelial injury in GSR is discussed.

Evidence for an essential role of tissue factor dependent blood coagulation in the pathogenesis of the local Shwartzman reaction.

Adherence and aggregation of leukocytes within the vessels of a prepared skin site has been shown to be essential for the pathogenesis of the local Shwartzman reaction (LSR) (Argenbright and Barton: J Clin Invest 89: 259, 1992). We have now performed experiments in rabbits to evaluate whether coagulation within the vessels of a prepared site is a second requirement for the reaction. Skin sites were prepared by an intradermal injection of endotoxin 24 hours before a provoking intravenous injection of endotoxin. Thirteen control rabbits all developed the LSR. Seven of 12 rabbits given warfarin to achieve anticoagulation approximating that used therapeutically in humans before the provoking injection were protected against the LSR (p = 0.003). Five of nine rabbits given anti-rabbit factor X IgG before the provoking injection to yield mean values in individual rabbits of between 7% and 18% plasma factor X activity were protected against the LSR (p = 0.009). Six of 11 rabbits given anti-rabbit factor VII IgG before the provoking injection to yield mean values in individual rabbits of between < 0.5% and 2.2% were protected against the LSR (p = 0.007). Four rabbits failed to develop the LSR at an endotoxin-prepared skin site when an infusion of tissue factor (TF) causing substantial intravascular coagulation was substituted for a provoking injection of endotoxin. It would appear that two events are required for the pathogenesis of the LSR provoked by endotoxin: formation of aggregated masses of WBC in the prepared skin vessels and deposition of fibrin due to TF-initiated coagulation.

The effect of SRI 63-675, a competitive platelet-activating factor receptor-antagonist, in the generalized Shwartzman reaction.

Evidence indicates a role for platelet-activating factor (PAF) in endotoxin (LPS)-induced shock. To determine its involvement in LPS-induced intravascular coagulation, we assessed the efficacy of SRI 63-675, a specific PAF receptor antagonist, to prevent fibrin deposition in the various organs of New Zealand White rabbits 4 h after two intravenous doses of LPS (100 micrograms/kg), spaced 24 h apart. SRI 63-675 significantly lowered peak tumor necrosis factor levels after LPS challenge. Administration of SRI 63-675 around either the first (150 mg/kg) or second LPS dose (120 mg/kg), however, did not prevent coagulation. The unexpected high mortality after combined administration of SRI 63-675 and LPS precluded assessment of PAF inhibition around both LPS doses on coagulation. Sole administration of SRI 63-675 induced rapid and transient changes in peripheral blood cell counts, and blood pressure and heart rate suggestive of intrinsic PAF-like activity. Although some other intrinsic toxic effect of SRI 63-675 cannot be ruled out, it is suggested that endotoxin may have primed the rabbit to the lethal, PAF-like, effects of SRI 63-675.

Protective effect of anti-interleukin (IL)-6 antibody against endotoxin, associated with paradoxically increased IL-6 levels.

Two rat monoclonal antibodies (6B4 and 20F3) against mouse interleukin (IL)-6 were studied for their effects on the generalized Shwartzman reaction and on cytokine production elicited by endotoxin injections. Both antibodies were found to protect mice against the generalized Shwartzman reaction. Production of interferon and tumor necrosis factor in these animals, as assessed from serum levels, were not consistently affected by the antibody treatment, although rather increased levels were occasionally noted. Paradoxically, however, endotoxin-induced serum levels of IL-6 in anti-IL-6-treated mice were consistently found to be markedly increased and also to persist for longer time periods. The more vigorous and persistent response may have been due to slower elimination, increased synthesis, or a combination of both. Endogenous production of IL-6 in mice may be sufficiently large to supersede the neutralizing potential of an excess of antibody, as was evident from the fact that ascites fluid of the anti-IL-6-producing 6B4 hybridoma was biologically active in the IL-6 assay.

Aminoglycosides modify the in vitro metachromatic reaction and murine generalized Shwartzman phenomenon induced by Salmonella minnesota R595 lipopolysaccharide.

Endotoxin-neutralizing activity may be an important property for antibiotics to be used in severe sepsis. Several antibiotics, belonging to different classes, were evaluated as to their endotoxin-neutralizing ability, using the inhibition of an in vitro metachromatic assay for lipopolysaccharides and a murine generalized Shwartzman reaction model. Gentamicin, amikacin, and sisomicin have been found to share significant in vitro antiendotoxin activity at an antibiotic/endotoxin ratio as low as 1.0/5 (by weight) and to reduce the murine generalized Shwartzman reaction at an antibiotic/endotoxin ratio of 3.3/5.

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.

Synthesis and characterization of gentiobiose heptaacetate conjugate vaccines that produce endotoxin-neutralizing antibodies.

We have prepared aminoethyl (AE), aminopropyl (AP), and aminopentyl (APT) derivatives of gentiobiose heptaacetate (GH). These spacer compounds (AEGH, APGH, APTGH) have been coupled to succinylated diphtheria toxoid (Suc.DT) to produce conjugate vaccines. These conjugates all bind to the anti-lipid A human monoclonal antibody A6(H4C5) in an ELISA binding assay. Rabbits immunized with the APGH conjugate vaccine in either Freund's complete adjuvant or aluminum hydroxide gel produced antibody levels of 5120 and 3600 ELISA units, respectively, compared to an antibody level of less than 20 ELISA units for the prebleed sera. Sera from mice immunized with either the aminopropyl or the aminopentyl conjugate had antibody levels of 5120 and 2560 ELISA antibody units, respectively. These antibodies neutralized endotoxin in a Limulus lysate neutralization assay. Protection against the local Shwartzman reaction was demonstrated (p less than 0.05) in eight out of nine rabbits immunized with the Suc-DT-APGH conjugate vaccine compared to three out of 10 rabbits immunized with the carrier protein Suc-DT. Passive transfer experiments demonstrated that four out of five rabbits receiving immune serum were protected from Shwartzman reaction compared to one out of five rabbits receiving normal serum (p less than 0.1). These results indicated that epitopes contained in gentiobiose heptaacetate when properly presented as conjugate vaccines were capable of inducing neutralizing antibodies against endotoxin.

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.

Ureteral blockade sensitizes to the generalized Shwartzman reaction.

Renal cortical necrosis (RCN) has been reported in the normal kidney of patients with a contralateral ureteral occlusion (UO). So far, studies have examined the mechanisms protecting the affected kidney from glomerular thrombosis and cortical necrosis; but to the authors' knowledge, none has ever investigated the potential role of UO on the occurrence of the associated disseminated intravascular coagulation (DIC) episode leading to RCN. Female rats with a ligature of the right or left ureter were given injections, at different times after surgery, of 400 micrograms Salmonella typhosa 0901 endotoxin. Other experimental groups included normal and sham-operation rats and animals with a unilateral nephrectomy or with one kidney rendered ischemic by complete ligature of the renal vessels and of the ureter. All the animals were sacrificed 4 hours after endotoxin, and kidney sections stained with PTAH were examined for the presence of fibrin thrombi. Glomerular thrombosis was never observed in any hydronephrotic kidney, but occurred with a low incidence (16%) in the contralateral organ in the group given endotoxin the second day after UO. The incidence and severity of glomerular capillary thrombosis gradually increased in the normal kidney as the delay between surgery and endotoxin was prolonged; the incidences (P less than 0.01) were 45% and 83%, respectively, after 6 and 10 days. Endotoxin failed totally to initiate the lesion 1 day after UO as well as in normal, sham-operation and unilaterally nephrectomized rats, and in animals with combined UO and ligature of the renal circulation. We conclude that the perfused hydronephrotic kidney liberates a factor(s) that sensitizes to DIC and glomerular thrombosis, typical of the generalized Shwartzman reaction.

Pharmacological studies of FUT-175, nafamstat mesilate. I. Inhibition of protease activity in in vitro and in vivo experiments.

FUT-175, 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate (nafamstat mesilate), a novel synthetic protease-inhibiting agent, was studied to determine its in vitro effects against various proteases and other enzymes, as well as to determine its in vivo protease inhibitory effects. FUT-175 was found to inhibit, in an intense, specific and reversible way, the enzyme activities of trypsin, C1r, C1s, thrombin, kallikrein and plasmin with IC50 values of the order of 10(-6)-10(-8) M. FUT-175 also inhibited complement-mediated hemolysis, including both classical and alternative pathways, sites of inhibition being on C1r and C1s as evidenced by the intermediate-cell technique. In animal model reactions in which the complement system is known to be involved as pathogenetic factors, e.g., Forssman shock, Forssman cutaneous vasculitis, zymosan-induced paw edema, endotoxin shock and local Shwartzman reaction, FUT-175 was highly effective in that, for example, intravenous dosing at 3 mg/kg could completely protect guinea pigs from the lethal Forssman shock. FUT-175 was also found to be effective in trypsin-induced shock in mice, in lethality due to thrombin-thrombosis in mice and in kinin formation in the inflammatory process in rats.

Plasma fibronectin concentration in animal models of sepsis and endotoxemia.

Plasma fibronectin is a nonspecific opsonin which mediates phagocytosis of particulate matter by macrophages. Fibronectin depletion results in depression of reticuloendothelial system phagocytic function. This may potentiate microvascular embolization and sludging in critical illness. It has been hypothesized that sepsis is a major cause of fibronectin depletion. To explore this hypothesis, plasma fibronectin concentrations were measured in rats with intraabdominal abscesses and in rabbits subjected to the generalized Shwartzman reaction (spaced doses of endotoxin). In both groups of animals there was a significant increase (P less than 0.05) rather than decrease in fibronectin concentrations at times when sepsis and disseminated intravascular coagulation were manifest. This study does not support the hypothesized relationship between sepsis and fibronectin depletion. Until the kinetics of fibronectin production and utilization are further delineated, caution must be exercised in the interpretation of immunoreactive plasma fibronectin levels.

Role of the acute phase response in the Shwartzman phenomenon.

Following elicitation of the local Shwartzman reaction by intradermal injection of Salmonella enteritidis lipopolysaccharide (LPS) in mice, there was a marked acute phase response which was monitored by measuring the serum levels of serum amyloid P component (SAP) and C3. Prednisolone therapy had no effect on either the cutaneous lesion or the accompanying acute phase response. Also, in vivo complement depletion with cobra factor did not affect the lesion or the SAP response despite gross reduction in serum C3 levels. In contrast, administration of colchicine at the same time as LPS suppressed both the acute phase response and the Shwartzman reaction. Inhibition of the cutaneous reaction by colchicine was abrogated by injecting mice with casein, and unrelated acute phase stimulus, the day before challenge with LPS. These observations suggest that acute phase proteins may participate in pathogenesis of the Shwartzman phenomenon.

Prostaglandins in the pathogenesis of the generalized Shwartzman reaction.

The GSR induced by a single injection of endotoxin in pregnant rats and cortisone-sensitized rabbits was prevented by bradykinin. The action is mediated by prostaglandins, such as evidenced by: (1) abolition of the effect of bradykinin by aspirin, (2) prevention of the Shwartzman reaction by infusions of PGA2 and PGE1, and (3) sensitization of the normal male rat to the generalized Shwartzman reaction by infusions of PGA2 and PGE1, and (3) sensitization of the normal male rat to the generalized Shwartzman reaction by indomethacin. The mechanism appears to be a neutralization of the essential alpha-adrenergic component of the generalized Shwartzman reaction.

Antiserum treatment of gram-negative bacteremia.

In order to lower the mortality rate from gram-negative bacteremia, 136 patients were treated with human antiserum against core glycolipid, or with control nonimmune human serum, in a double-blind clinical trial. The antiserum was prepared by immunizing healthy young men with a vaccine composed of heat killed cells of the J5 mutant of E. coli 0111 B4. Since the core glycolipid in this mutant is not encumbered with "O" side chains, it can stimulate antibody against the core glycolipid possessed in common by the different species of gram-negative bacteria responsible for lethal bacteremia in patients. No serious reactions occurred in over 300 men receiving this vaccine, and the J5 antiserum gave striking broad-spectrum protection against experimental gram-negative bacteremia and endotoxemia. When human J5 antiserum was administered to seriously ill bacteremic patients, the mortality rate was virtually cut in half, as compared to controls. The death rate from gram-negative bacteremia was 14% in patients treated with J5 antiserum, and 26% in those given nonimmune control human serum. Among patients in profound gram-negative bacteremic shock, the recovery rate rose from 29% in controls to 82% in those treated with J5 antiserum (p = 0.02). On the basis of these encouraging results we propose to treat more bacteremic patients with J5 antiserum, because larger groups are needed to establish the full significance of the initial findings.

Prevention by aspirin of the classic generalized Shwartzman reaction.

The classic generalized Shwartzman reaction induced in the rabbit was prevented with large doses of aspirin (250 mg/kg) when administered at the time of the first and preparing injection of endotoxin. Such a result was not observed when the drug was given at the time of the second and provoking injection of endotoxin. Our investigations on platelet and coagulation indicate that aspirin prevents disseminated intravascular coagulation through an interference with the blood coagulation and Hageman factor activation rather than by the inhibition of platelet aggregation and availability of platelet procoagulant activity.

The effects of indomethacin on the generalized shwartzman reaction.

The antiflammatory drug indomethacin, an inhibitor of prostaglandin synthesis, prevents the generalized Shwartzman reaction produced in rabbits by two intravenous injections of bacterial endotoxin. Indomethacin has this effect if given before the first but not the second injection of endotoxin. Measurements of circulating white blood cells, platelets, partial thromboplastin time, prothrombin time, fibrinogen, plasminogen, and soluble fibrin were made at several times after either the first or second injection of endotoxin treated and nontreated rabbits. Four hours after the first injection of endotoxin, leukopenia and thrombocytopenia were somewhat greater in treated rabbits and the prolongation of the activated partial thromboplastin time was shortened. Twenty-one hours after injection of endotoxin, leukocytosis and elevation of plasma fibrinogen were not as great in treated animals. Four hours following the second injection of endotoxin a decrease in fibrinogen, prolongation of the prothrombin time, and the elaboration of soluble fibrin were consistently found in rabbits with the generalized Shwartzman reaction. In treated rabbits, none of these changes occurred. Indomethacin prevents the generalized Shwartzman reaction by preventing the development of the prepared state in this endotoxin model.