Immune Mechanisms Underlying Susceptibility to Endotoxin Shock in Aged Hosts: Implication in Age-Augmented Generalized Shwartzman Reaction.

In recent decades, the elderly population has been rapidly increasing in many countries. Such patients are susceptible to Gram-negative septic shock, namely endotoxin shock. Mortality due to endotoxin shock remains high despite recent advances in medical care. The generalized Shwartzman reaction is well recognized as an experimental endotoxin shock. Aged mice are similarly susceptible to the generalized Shwartzman reaction and show an increased mortality accompanied by the enhanced production of tumor necrosis factor (TNF). Consistent with the findings in the murine model, the in vitro Shwartzman reaction-like response is also age-dependently augmented in human peripheral blood mononuclear cells, as assessed by enhanced TNF production. Interestingly, age-dependently increased innate lymphocytes with T cell receptor-that intermediate expression, such as that of CD8+CD122+T cells in mice and CD57+T cells in humans, may collaborate with macrophages and induce the exacerbation of the Shwartzman reaction in elderly individuals. However, endotoxin tolerance in mice, which resembles a mirror phenomenon of the generalized Shwartzman reaction, drastically reduces the TNF production of macrophages while strongly activating their bactericidal activity in infection. Importantly, this effect can be induced in aged mice. The safe induction of endotoxin tolerance may be a potential therapeutic strategy for refractory septic shock in elderly patients.

Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation.

Endothelial cell activation plays a critical role in regulating leukocyte recruitment during inflammation and infection. Based on recent studies showing that acetylcholine and other cholinergic mediators suppress the production of proinflammatory cytokines via the alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expressed by macrophages and our observations that human microvascular endothelial cells express the alpha7 nAChR, we examined the effect of cholinergic stimulation on endothelial cell activation in vitro and in vivo. Using the Shwartzman reaction, we observed that nicotine (2 mg/kg) and the novel cholinergic agent CAP55 (12 mg/kg) inhibit endothelial cell adhesion molecule expression. Using endothelial cell cultures, we observed the direct inhibitory effects of acetylcholine and cholinergic agents on tumor necrosis factor (TNF)-induced endothelial cell activation. Mecamylamine, an nAChR antagonist, reversed the inhibition of endothelial cell activation by both cholinergic agonists, confirming the antiinflammatory role of the nAChR cholinergic pathway. In vitro mechanistic studies revealed that nicotine blocked TNF-induced nuclear factor-kappaB nuclear entry in an inhibitor kappaB (IkappaB)alpha- and IkappaBepsilon-dependent manner. Finally, with the carrageenan air pouch model, both vagus nerve stimulation and cholinergic agonists significantly blocked leukocyte migration in vivo. These findings identify the endothelium, a key regulator of leukocyte trafficking during inflammation, as a target of anti-inflammatory cholinergic mediators.

Resistance of natural killer T cell-deficient mice to systemic Shwartzman reaction.

The generalized Shwartzman reaction in mice which had been primed and challenged with lipopolysaccharide (LPS) depends on interleukin (IL)-12-induced interferon (IFN)-gamma production at the priming stage. We examined the involvement in the priming mechanism of the unique population of Valpha14 natural killer T (NKT) cells because they promptly produce IFN-gamma after IL-12 stimulation. We report here that LPS- or IL-12-primed NKT cell genetically deficient mice were found to be resistant to LPS-elicited mortality. This outcome can be attributed to the reduction of IFN-gamma production, because injection of recombinant mouse IFN-gamma, but not injection of IL-12, effectively primed the NKT cell-deficient mice. However, priming with high doses of LPS caused mortality of severe combined immunodeficiency, NKT cell-deficient, and CD1-deficient mice, indicating a major contribution of NKT cells to the Shwartzman reaction elicited by low doses of LPS, whereas at higher doses of LPS NK cells play a prominent role. These results suggest that the numerically small NKT cell population of normal mice apparently plays a mandatory role in the priming stage of the generalized Shwartzman reaction.

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.

Whatever happened to the Shwartzman phenomenon?

Ninety years ago, Gregory Shwartzman first reported an unusual discovery following the intradermal injection of sterile culture filtrates from principally Gram-negative strains from bacteria into normal rabbits. If this priming dose was followed in 24 h by a second intravenous challenge (the provocative dose) from same culture filtrate, dermal necrosis at the first injection site would regularly occur. This peculiar, but highly reproducible, event fascinated the microbiologists, hematologists, and immunologists of the time, who set out to determine the mechanisms that underlie the pathogenesis of this reaction. The speed of this reaction seemed to rule out an adaptive, humoral, immune response as its cause. Histopathologic material from within the necrotic center revealed fibrinoid, thrombo-hemorrhagic necrosis within small arterioles and capillaries in the micro-circulation. These pathologic features bore a striking resemblance to a more generalized coagulopathic phenomenon following two repeated endotoxin injections described 4 yr earlier by Sanarelli. This reaction came to be known as the generalized Shwartzman phenomenon, while the dermal reaction was named the localized or dermal Shwartzman reaction. A third category was later added, called the single organ or mono-visceral form of the Shwartzman phenomenon. The occasional occurrence of typical pathological features of the generalized Shwartzman reaction limited to a single organ is notable in many well-known clinical events (e.g., hyper-acute kidney transplant rejection, fulminant hepatic necrosis, or adrenal apoplexy in Waterhouse-Fredrickson syndrome). We will briefly review the history and the significant insights gained from understanding this phenomenon regarding the circuitry and control mechanisms responsible for disseminated intravascular coagulation, the vasculopathy and the immunopathy of sepsis.

Human antiserum for prevention of the local shwartzman reaction and death from bacterial lipopolysaccharides.

Bacterial lipopolysaccharides from dead bacteria have been blamed for the continuing high mortality from gram-negative infections despite antibiotic treatment. Because animal antiserum against these lipopolysaccharides has been shown to protect against several of the effects of endotoxin, we undertook the development of antiserum in human subjects. 21 men were immunized with a single injection of Salmonclla typhimurium or Escherichia coli 0:111 heat-killed cells and immune serum was collected at 2 wk. Preimmune serum was obtained as a control in all animal experiments. 1 ml antiserum given intravenously protected mice against a lethal intravenous dose of homologous endotoxin (P < 0.005 for both antisera). E. coli antiserum reduced the incidence of positive local Shwartzman reactions with E. coli endotoxin from 100 to 38%; S. typhimurium antiserum reduced the incidence from 92 to 35%. (P < 0.0005 for both antisera). There was no protection against heterologous endotoxin in either animal model. These experiments demonstrate for the first time that human antiserum confers exceedingly potent passive immunity to the effects of endotoxin.

Mice deficient in Vbeta8+NKT cells are resistant to experimental hepatitis but are partially susceptible to generalised Shwartzman reaction.

NKT cells are responsible for hepatitis induced either by concanavalin A (Con-A) or alpha-galactosylceramide (alpha-GalCer), and they are also profoundly involved in the generalised Shwartzman reaction (GSR) induced by consecutive injections of interleukin (IL)-12 and lipopolysaccharide (LPS). In the present study, using NC/Nga (NC) mice and SJL mice lacking the Vbeta(+)8 gene, we examined the role of Vbeta(+)8+NKT cells in hepatitis models and in the GSR. The absence of Vbeta(+)8+NKT cells in the liver mononuclear cells (MNC) was confirmed by the alpha-GalCer/CD1d/Ig dimer. Unexpectedly, other dimer+NKT cells including Vbeta7(+)NKT cells in these mice were found to decrease in comparison to that of C57BL/6 mice. No significant hepatocyte injury was observed after alpha-GalCer or Con-A administration in either mice. The serum interferon (IFN)-gamma, IL-4 and tumour necrosis factor (TNF) levels did not increase in these mice after alpha-GalCer injection, however these cytokines substantially increased after Con-A administration, thus suggesting that the roles of NKT cells differ between the two hepatitis models. However, in GSR, although neither mice showed lower IFN-gamma levels after a priming IL-12 injection, they showed TNF levels comparable to those in normal mice after LPS injection, and thus resulted in a decreased but substantial mortality. Although liver MNC from IL-12-injected SJL mice showed an impaired antitumour cytotoxicity, liver MNC of NC mice exhibited a greater antitumour cytotoxicity than that of C57BL/6 mice because liver NK cells proportionally increased in NC mice. These results confirm the critical role that Vbeta8(+)NKT cells play in both liver and multi-organ injury.

An in vitro Shwartzman reaction-like response is augmented age-dependently in human peripheral blood mononuclear cells.

A lethal human septic shock model, mouse generalized Shwartzman reaction (GSR), was elicited by two consecutive lippolysaccharide (LPS) injections (24 h apart) in which interferon-gamma (IFN-gamma) induced by interleukin (IL)-12 played a critical role in the priming phase, and tumor necrosis factor (TNF) was an important effector molecule in the second phase. We recently reported IL-12/LPS-induced mouse GSR age-dependently enhanced. We herein demonstrate that human peripheral blood mononuclear cells (PBMC) from healthy adults/elderly, cultured with IL-12 for 24 h and with LPS for an additional 24 h, produced a much larger amount of TNF (which increased age-dependently) than did PBMC without IL-12 priming. Whereas macrophages mainly produced TNF following LPS stimulation, macrophages and lymphocytes were necessary for a sufficient TNF production. IL-12-induced IFN-gamma up-regulated Toll-like receptor 4 (TLR-4) on macrophages of adults. Although the PBMC from children produced a substantial amount of IFN-gamma after IL-12 priming, the GSR response, with augmented TNF production and an up-regulated TLR-4 expression of macrophages, was not elicited by LPS stimulation. CD56+natural killer cells, CD56+T cells, and CD57+T cells (NK-T cells), which age-dependently increased in PBMC, produced much larger amounts of IFN-gamma after IL-12 priming than that of conventional CD56-CD57-T cells and also induced cocultured macrophages to produce TNF by subsequent LPS stimulation. The elder septic patients were consistently more susceptible to lethal shock with enhanced serum TNF levels than the adult patients. The NK cells, NK-T cells, and macrophages, which change proportionally or functionally with aging, might be involved in the enhanced GSR response/septic shock observed in elderly patients.

Brief Report: Endothelial-Specific X-Box Binding Protein 1 Deficiency Limits Tumor Necrosis Factor-Induced Leukocyte Recruitment and Vasculitis.

OBJECTIVE: Endothelial cell activation by tumor necrosis factor (TNF) and associated leukocyte infiltration are hallmarks of vasculitis. The aim of this study was to investigate the potential role of the cellular stress-associated endothelial X-box binding protein 1 (XBP-1) transcription factor in TNF-induced endothelial cell inflammation and vasculitis. METHODS: Mice with an endothelial cell-specific XBP-1 deficiency were used in a modified local Shwartzman reaction (LSR) model of TNF-induced small vessel vasculitis. To address the contribution of XBP-1 to the TNF-mediated inflammatory response in endothelial cells, we examined the activation of XBP-1 expression by TNF as well as the effect of XBP-1 knockdown in endothelial cells on TNF-induced signaling, proinflammatory gene expression, and leukocyte-endothelial cell adhesion. RESULTS: The active spliced form of XBP-1 in endothelial cells was triggered by TNF. In addition, endothelial XBP-1 contributed to the sustained TNF-triggered NF-κB-dependent transcriptional activation of proinflammatory molecules, which was associated with leukocyte-endothelial cell adhesion. In the LSR model, endothelial cell-specific XBP-1-deficient mice displayed significantly less vascular damage, accompanied by reduced perivascular neutrophil infiltration, as compared with wild-type mice. CONCLUSION: Endothelial XBP-1 is activated by TNF and regulates leukocyte-endothelial cell adhesion in vitro as well as neutrophil infiltration and vascular damage in murine vasculitis.

Effect of soluble P55 tumour-necrosis factor binding fusion protein on the local Shwartzman and Arthus reactions.

1. In this study, the effects of a protein synthesis inhibitor, cycloheximide, and a soluble tumour necrosis factor (TNF) binding/IgG fusion protein, p55-sf2, on the priming and challenge stages of the local Shwartzman reaction (LSR) were assessed and compared with their effects on the acute inflammatory response induced by recombinant human tumour necrosis factor-alpha (rhTNF), lipopolysaccharide (LPS) and a reversed passive Arthus (RPA) reaction in rabbit skin. 2. The LSR was induced in skin by giving an intradermal (i.d.) priming injection of LPS followed by two i.v. challenge injections 20 h and 22 h later. Accumulation of 51-Cr-labelled red blood cells and [125I]-albumin were measured at 24 h as markers of haemorrhage and oedema formation, respectively. 3. The RPA reaction was induced in the rabbit by giving i.d. injections of Arthus anti-serum (anti-bovine-gamma-globulin, BGG) followed 5 min later by an i.v. injection of the antigen (BGG). Oedema formation and the accumulation of 111In-labelled neutrophils produced in the RPA reaction and in response to i.d. injection of rhTNF and LPS were measured over the 4 h period after inducing the responses. 4. A single local injection of cycloheximide (10 micrograms/site) did not inhibit neutrophil accumulation or oedema formation produced by 100% Arthus anti-sera. Although LPS injected i.d. induced a marked dose-dependent neutrophil accumulation, there was little associated plasma leakage. Cycloheximide (10 micrograms/site) did not significantly inhibit the neutrophil accumulation induced by LPS (0.1 microgram/site). In the LSR, priming i.d. injections of LPS caused a dose-dependent increase in haemorrhage and plasma leakage at skin sites after challenge with LPS (two injections of 100 micrograms, i.v.). Co-injection of a single dose of cycloheximide (10 micrograms/site) with LPS (30 micrograms/site) caused a marked reduction in the amount of haemorrhage. Local cycloheximide (10 micrograms/site) administered immediately before LSR challenge did not affect the responses produced in the LSR. 5. Neutrophil accumulation induced by TNF (0.17 micrograms/site) was abolished by co-administration of p55-sf2 (3 micrograms/site) whereas neutrophil accumulation induced by i.d. LPS and produced in the RPA reaction was not affected. In the LSR, haemorrhage and oedema formation were inhibited by p55-sf2 (3 micrograms/site) when it was administered i.d. with the LPS priming injection, but not when given i.d. immediately before LSR challenge. 6. These data suggest that the acute neutrophil accumulation produced in the RPA reaction and in response to i.d. LPS may not be dependent on local protein synthesis or TNF production. On the other hand, haemorrhage appears to be dependent on local protein synthesis during the priming phase but not during the challenge stage of the LSR. Importantly, haemorrhage and plasma leakage appear to be dependent on local TNF generation during the priming phase but not during the challenge stage of the LSR. Thus TNF appears to play a key role in the LSR in rabbit skin.

Attempted induction of local Shwartzman reaction in the chicken.

Chickens and rabbits were injected intradermally with an endotoxin, namely Escherichia coli lipopolysaccharide (LPS). Twenty-four hours later, LPS was again administered intravenously to induce a local Shwartzman reaction. A typical cutaneous inflammatory reaction developed in rabbits, but not in chickens. Even very high doses of LPS, that made the birds visibly sick, failed to elicit the reaction. The results suggest that chickens are refractory to the Shwartzman reaction. A noteworthy feature of the chickens' response to intradermal endotoxin was the formation of prominent perivascular lymphoid aggregates.

[An immunological study of the cell wall proteins of Pseudomonas aeruginosa]. / Immunologicheskoe izuchenie belkov kletochnoi stenki Pseudomonas aeruginosa.

A preparation containing P. aeruginosa cell-wall proteins with a mol. wt. of 9-55 kD has been obtained from P. aeruginosa by the method of extraction with the use of tris-EDTA buffer. In experiments on mice this protein preparation has shown pronounced protective properties, but, according to the data of Shwartzman's local reaction, proved to be toxic, perhaps due to the admixture of LPS. The purification of the protein preparation from the admixture of LPS will make it possible to obtain an effective vaccine against P. aeruginosa infection.

The effect of synthetic C-reactive protein on the in vitro immune response of human PBMCs stimulated with bacterial reagents.

Synthetic C-reactive protein (CRP) rescues mice from lethal endotoxin shock or bacterial infection by suppressing tumor necrosis factor (TNF-α), but in turn, enhances Kupffer cell phagocytic activity. We herein assessed the influence of CRP in human peripheral blood mononuclear cells (PBMCs). When human PBMCs were stimulated in vitro with penicillin-treated Streptococcus pyogenes, bacterial DNA motifs and lipopolysaccharide with or without synthetic CRP, CRP suppressed the production of TNF-α and IL-12, but not that of IFN-γ. This was also the case for the in vitro Shwartzman reaction induced in PBMCs. CRP also decreased high-mobility group box 1 production from macrophages, which is crucial in the later phase of endotoxin/septic shock. However, CRP upregulated the perforin expression by CD56(+) NK cells and increased their antitumor cytotoxicity. CRP may thus be a potent immunomodulatory factor in the human immune system, suggesting its therapeutic potential for use against human septic shock.