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.

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.

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.

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.

Interferon-gamma: a historical perspective.

This article reviews the main lines of thinking and exploration that have led to our current conception of the role of IFN-gamma in immune defense and autoimmunity. In 1965 the first report appeared describing production of an interferon-like virus inhibitor in cultured human leukocytes following exposure to the mitogen phytohemagglutinin. In the early 1970s the active principle became recognized as being distinct from classical virus-induced interferons, leading to its designation as immune interferon or Type II interferon, and eventually IFN-gamma. Up to that point interest in the factor had come almost exclusively from virologists, in particular those among them who were believers in interferon. Evidence first coming forward in the 1980s that IFN-gamma is indistinguishable from macrophage-activating factor (MAF), then a prototype lymphokine, was the signal for immunologists at large to become interested. Today IFN-gamma ranks among the most important endogenous regulators of immune responses.

Requirement for Vav proteins in post-recruitment neutrophil cytotoxicity in IgG but not complement C3-dependent injury.

The signals linking neutrophil opsonic receptors, FcgammaRs and complement receptor 3 (Mac-1) to cellular cytotoxic responses are poorly understood. Furthermore, because a deficiency in activating FcgammaRs reduces both IgG-mediated neutrophil recruitment and tissue injury, the role of FcgammaRs specifically in mediating neutrophil cytotoxicity in vivo remains unclear. In this study, we demonstrate that neutrophil Vav 1 and 3, guanine exchange factors for Rac GTPases, are required for IgG/FcgammaR-mediated hemorrhage and edema in the reverse passive Arthus in the lung and skin. Rac GTPases are also required for development of the reverse passive Arthus reaction. A deficiency in Vav 1 and 3 does not affect neutrophil accumulation at the site of immune complex deposition, thus uncoupling neutrophil recruitment and tissue injury. Surprisingly, Vav and Rac proteins are dispensable for the development of the local Shwartzman reaction in vivo and phagocytosis of complement-opsonized RBC in vitro, processes strictly dependent on Mac-1 and complement C3. Thus, FcgammaR signaling through the Vav and Rac proteins in neutrophils is critical for stimulating immune complex disease while Vav- and Rac-independent pathways promote Mac-1/complement C3-dependent functions.

Galectin-9 protects mice from the Shwartzman reaction by attracting prostaglandin E2-producing polymorphonuclear leukocytes.

Galectins play a crucial role in the modulation of innate and adaptive immunity. Here we show that galectin-9 (Gal-9) exhibits an anti-inflammatory role in LPS-induced inflammation. Intraperitoneal LPS injection enhances Gal-9 levels as well as promotes the production of pro-inflammatory cytokines, e.g., TNF-alpha, IFN-gamma and IL-12. We found that Gal-9 administration results in the protection of mice from the Shwartzman reaction, and Gal-9-deficient mice became susceptible to the Shwartzman reaction, thus implying the anti-inflammatory activity of Gal-9 against LPS-induced inflammation. Indeed, Gal-9 treatment together with LPS suppresses production of these pro-inflammatory cytokines, while it rather enhances than suppresses IL-4 and IL-10 production. We also found that LPS-induced elevation of TNF-alpha, IFN-gamma, and IL-12 does not occur in Gal-9 transgenic mice. Moreover, Gal-9 induces Gr-1(+) cell; probably polymorphonuclear leukocyte (PMN), as well as infiltration in to the peritoneal cavity, causing us to hypothesize PMNs are involved in Gal-9-mediated suppression. The fact that Gal-9 does not suppress LPS-induced TNF-alpha, IFN-gamma and IL-12 production in neutropenic mice, and that it does not protect those mice from the Shwartzman reaction, confirms the involvement of PMN in regulation. PMN attracted by Gal-9 produce PGE(2), which LPS-induced TNF-alpha production from the peritoneal macrophages is suppressed, while PMNs attracted by casein produce less PGE(2) and fail to suppress LPS-induced TNF-alpha production. Our data suggest that Gal-9 regulates LPS-induced inflammation and protects mice from the Shwartzman reaction by attracting PGE(2)-producing PMN.

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.

Loss of SLP-76 expression within myeloid cells confers resistance to neutrophil-mediated tissue damage while maintaining effective bacterial killing.

The Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is an adaptor molecule critical for immunoreceptor and integrin signaling in multiple hemopoietic lineages. We showed previously that SLP-76 is required for neutrophil function in vitro, including integrin-induced adhesion and production of reactive oxygen intermediates, and to a lesser extent, FcgammaR-induced calcium flux and reactive oxygen intermediate production. It has been difficult to determine whether SLP-76 regulates neutrophil responses in vivo, because Slp-76(-/-) mice exhibit marked defects in thymocyte and vascular development, as well as platelet and mast cell function. To circumvent these issues, we generated mice with targeted loss of SLP-76 expression within myeloid cells. Neutrophils obtained from these animals failed to respond to integrin activation in vitro, similar to Slp-76(-/-) cells. Despite these abnormalities, SLP-76-deficient neutrophils migrated normally in vivo in response to Staphylococcus aureus infection and efficiently cleared micro-organisms. Interestingly, SLP-76-deficient neutrophils did not induce a robust inflammatory response in the localized Shwartzman reaction. Collectively, these data suggest that disruption of integrin signaling via loss of SLP-76 expression differentially impairs neutrophil functions in vivo, with preservation of migration and killing of S. aureus but reduction in LPS-induced tissue damage and vascular injury.

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.

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.

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.

Critical role of the liver CD8+ CD122+ T cells in the generalized Shwartzman reaction of mice.

We examined the role of mouse CD8+ CD122+ T cells, which increase in number with age, in the generalized Shwartzman reaction. This reaction was induced by IL-12 priming and subsequent LPS challenge (after 24 h) in mice of various ages (4-50 weeks of age). Although most young mice (4 or 6 weeks of age) survived, mortality essentially increased with increasing age of the mice, and all mice of 20 weeks of age or older died within 48 h. Serum TNF-alpha levels after LPS challenge also increased age dependently. The neutralization of either IL-12-induced IFN-gamma or LPS-induced TNF-alpha improved the survival of middle-aged (25-week-old) mice. Both IFN-gamma production after IL-12 priming and TNF-alpha production from the liver mononuclear cells after LPS challenge were also prominent in the middle-aged mice. CD8+CD122+ T cells cultured with IL-12 produced a much larger amount of IFN-gamma than CD8+CD122- T cells. Although the depletion of NK/NK T cells did not decrease the IFN-gamma or TNF-alpha production in the Shwartzman reaction of the middle-aged mice, an additional depletion of CD8+CD122+ T cells did decrease such production and also improved mouse survival. Furthermore, young mice transferred with CD8+CD122+ T cells from aged B6 nude mice showed an enhanced Shwartzman reaction.

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.

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.

The potential role of the Arthus and Shwartzman reactions in the pathogenesis of pneumonic pasteurellosis.

Pneumonic pasteurellosis (PP) is an economically important disease in cattle, sheep, and goats. Pasteurella haemolytica is commonly isolated from the severe fibrinopurulent pneumonia that characterize this respiratory syndrome. During infection, the bacteria produce leukotoxin (LKT) and lipopolysaccharide (LPS), both potent inducers of inflammation. Nonetheless, it has also been demonstrated that an exacerbated host's inflammatory response is responsible for the severe lung damage. Despite research in this field, the pathogenesis of PP is still incomplete. Two classical models of acute inflammatory response induced in laboratory animals, the Arthus and Shwartzman reactions, could explain the pathogenesis of the severe lung lesions that characterize PP.

The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses.

The liver remains a hematopoietic organ after birth and can produce all leukocyte lineages from resident hematopoietic stem cells. Hepatocytes produce acute phase proteins and complement in bacterial infections. Liver Kupffer cells are activated by various bacterial stimuli, including bacterial lipopolysaccharide (LPS) and bacterial superantigens, and produce interleukin (IL)-12. IL-12 and other monokines (IL- 18 etc.) produced by Kupffer cells activate liver natural killer (NK) cells and NK1.1 Ag+ T cells to produce interferon-gamma and thereby acquire cytotoxicity against tumors and microbe-infected cells. These liver leukocytes and the T helper 1 immune responses induced by them thus play a crucial role in the first line of defense against bacterial infections and hematogenous tumor metastases. However, if this defense system is inadequately activated, shock associated with multiple organ failure takes place. Activated liver NK1.1 Ag+ T cells and NK cells also cause hepatocyte injury. NK1.1 Ag+ T cells and another T-cell subset with an intermediate T-cell receptor, CD 122+CD8+ T cells, can develop independently of thymic epithelial cells. Liver NK cells and NK1.1 Ag+ T cells physiologically develop in situ from their precursors, presumably due to bacterial antigens brought from the intestine via the portal vein. NK cells activated by bacterial superantigens or LPS are also probably involved in the vascular endothelial injury in Kawasaki disease.

Cutting edge: IL-15 costimulates the generalized Shwartzman reaction and innate immune IFN-gamma production in vivo.

Sequential administration of LPS to SCID mice results in the generalized Shwartzman reaction, manifesting as rapid mortality via cytokine-induced shock. Here we demonstrate that in vivo neutralization of IL-15 before LPS priming significantly reduced lethality in this reaction (p = 0.0172). We hypothesize that LPS priming induces IL-12 and IL-15 that costimulate NK cell-derived IFN-gamma. Such IFN-gamma may then in turn sensitize macrophages to elicit the Shwartzman reaction following a subsequent LPS challenge. Supporting this, IL-12 and IL-15 synergized to induce murine NK cell IFN-gamma production in vitro. LPS stimulation of SCID mouse splenocytes resulted in measurable IFN-gamma production, which was reduced when IL-15 was neutralized or IL-2/15Rbeta was blocked. Pretreatment with either anti-IL-2/15Rbeta or anti-IL-15 Abs reduced serum IFN-gamma protein following LPS administration to SCID mice. Collectively, these data provide the first in vivo evidence that IL-15 participates in LPS-induced innate immune IFN-gamma production and significantly contributes to the lethal Shwartzman reaction.