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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.

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.

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., (© 2015, American College of Rheumatology.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Previously we reported that the consecutive injection of lipopolysaccharide (LPS) into LPS-sensitized mice for the generalized Shwartzman reaction (GSR) appeared to induce the injury of renal tubular epithelial cells via apoptosis. The aim of this study was to characterize the mechanism of renal tubular epithelial cell injury in GSR. The expression of Fas and Fas ligand was immunohistochemically detected on renal tubular epithelial cells from GSR-induced mice, although neither Fas nor Fas ligand was found in cells from untreated control mice or in cells from mice receiving a single injection of LPS. GSR-induced renal tubular epithelial cell injury was produced in neither Fas-negative MRL-lpr/lpr mice nor Fas ligand-negative MRL-gld/gld mice. The administration of anti-gamma interferon antibody together with a preparative injection of LPS prevented the expression of Fas and Fas ligand and the apoptosis of renal tubular epithelial cells. A provocative injection of tumor necrosis factor alpha into LPS-sensitized mice augmented Fas and Fas ligand expression and the apoptosis of renal tubular epithelial cells. The administration of tumor necrosis factor alpha to interleukin-12-sensitized mice resulted in Fas and Fas ligand expression and the apoptosis. Sensitization with interleukin-12 together with anti-gamma interferon antibody did not cause the apoptosis of renal tubular epithelial cells. It was suggested that the Fas/Fas ligand system probably plays a critical role in the development of renal tubular epithelial cell injury through apoptotic cell death.

Vascular injury in vasculitis may be due to activation of circulating neutrophils resulting in their increased adhesiveness to locally activated endothelium (Shwartzman phenomenon). Previously, we demonstrated up-regulation of endothelial intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in biopsies from patients with ANCA-associated vasculitis. In the present study, we investigated the expression of adhesion molecules (CD11b, ICAM-1, VLA-4, L-selectin) and activation markers (CD66b, CD64, CD63) on circulating neutrophils from patients with ANCA-associated vasculitis in comparison with their expression on cells from healthy volunteers and patients with sepsis. We related these findings to parameters of disease activity. Surface marker expression was determined by using a non-activating whole blood flow cytometric assay. The expression of activation markers, but not the expression of adhesion molecules, was increased on neutrophils from patients with active vasculitis. The expression of CD63 and CD66b on neutrophils correlated with disease activity as determined by the Birmingham Vasculitis Activity Score (BVAS). In contrast to patients with active vasculitis, patients with sepsis showed up-regulation of all markers, including adhesion molecules, suggesting that circulating neutrophils are fully activated in sepsis. We conclude that in ANCA-associated vasculitis, circulating neutrophils are not fully activated, since they do not express increased levels of adhesion molecules as sepsis or in the Shwartzman reaction. These findings are compatible with the concept that in vivo vascular damage in ANCA-associated vasculitides does not occur due to a Shwarzman-like reaction but only after ANCA-induced neutrophil activation at the endothelial cell surface.

IL-12 (or LPS) priming and subsequent challenge by LPS produces the generalized Shwartzman reaction. IFN-gamma induced by IL-12 is a crucial cytokine in the priming phase. In vivo depletion of both NK cells and NK1+ alphabeta T cells of mice by anti-NK1.1 Ab greatly reduced the elevation of serum IFN-gamma induced by IL-12 and significantly reduced mortality after subsequent injection of LPS, whereas depletion of NK cells alone by anti-asialo GM1 Ab only partially decreased serum IFN-gamma, and lethality was not changed. Cell sorting and culture experiments confirmed that liver NK1+ alphabeta T cells of IL-12-injected mice produced greater amounts of IFN-gamma than did liver NK cells. MHC class I-deficient mice of C57BL/6 background, which lack a majority of NK1+ alphabeta T cells, produced low amounts of IFN-gamma by IL-12; no mortality was observed after the LPS challenge. However, production of TNF-alpha in the second phase (after LPS challenge) was not inhibited by depletion of NK cells alone or both subsets. IL-12 and subsequent LPS challenge activated NK1+ alphabeta T cells in the liver and induced strong cytotoxicity of these cells not only against tumor cells (including Fas-negative tumors) but also against a syngeneic hepatocyte cell line. Our findings show that IFN-gamma produced by NK1+ alphabeta T cells is essential for the IL-12 priming of the Shwartzman reaction, and the autoreactivity of NK1+ alphabeta T cells in the liver is involved in the hepatic disorders that are sometimes caused by IL-12, LPS, or the generalized Shwartzman reaction.

A local Shwartzman reaction (LSR) was prepared in rabbit lung as a model of acute lung injury. To induce LSR, intratracheal injection of lipopolysaccharide (LPS) 10 micrograms into the lower lobe of the right lung, followed 24 h later by i.v. injection of LPS (10 micrograms/kg). In the lung with the LSR, myeloperoxidase activity, representing neutrophil accumulation, peaked at 1-2 h and was sustained for 48 h after challenge with i.v. LPS. The lung water content peaked at 12 h, and decreased gradually. Histological findings showed diffuse interstitial widening, intra-alveolar leukocyte infiltration with hemorrhage, and alveolar exudate formation. The production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8), and IL-1 receptor antagonist (IL-1 Ra) in the lung was analyzed. TNF-alpha first elevated and peaked at 0.5 h (66.5 +/- 16.7 ng/g.lung), subsequently, IL-1 beta and IL-8 increased and peaked at 2 h (17.8 +/- 3.4 ng/g.lung and 336.9 +/- 49.6 ng/g.lung, respectively). IL-1Ra was present even before the challenge, and the production increased to show a dual peak (0.5 h, 1.5 +/- 0.2 micrograms/g.lung; and 2 h, 1.6 +/- 0.1 micrograms/g.lung), and a large concentration of IL-1Ra was sustained for 48 h. Immunohistochemistry showed that the cellular source of these cytokines was alveolar macrophages and infiltrating neutrophils. Thus, disclosing the kinetics of the generation of cytokines led to a better understanding of their roles, namely TNF-alpha as an initiator, IL-1 and IL-8 as amplifier and effector, and IL-1Ra as regulator of the intensity of acute inflammation.

Immunologically, the septic shock is a natural model of immunomediated vascular pathology where the interaction between cytokines and the endothelium mediates the syndrome and lethality. Tumour necrosis factor (TNF), a non-species-specific cytokine, has outstanding pleiotropic activities as an important mediator of the septic shock syndrome. In rabbits, passive immunization with anti-lipopolysaccharide (LPS) polyclonal antibodies prior to the intravenous (i.v.) injection of LPS inhibits the haemorrhagic necrotic lesion characteristic of the local Shwartzman reaction (an excellent localized in vivo correlate of the septic shock). Paradoxically, tested in an ex vivo assay (short-term whole human blood culture, stimulated with LPS), these antibodies mediated an increase in TNF production by mononuclear phagocytes and, in the rabbit model, they induced an increase in body temperature, as compared with the pre-immune reagent. Although anchoring of immune complexes containing LPS to receptors (Fc or C4b-C3b) on circulating monocytes may facilitate the access of LPS to these cells, access to localized, LPS-sensitized macrophages may be impaired. Consequently inhibition of the local Shwartzman reaction and increased TNF production in the ex vivo system were observed. Concordantly, the higher temperature in the passively immunized animals may be a consequence of a higher, immune complex-induced, systemic TNF production. These experimental results suggest that the use of anti-LPS immunoglobulins, as a potential immunotherapy for septic shock syndrome in vertebrates, may lead to increased TNF production, with adverse effects such as the pyrogenic.

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.

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.

Observations in our laboratory have provided evidence that interferon-gamma (IFN-gamma) is a key regulator of inflammatory responses to bacterial lipopolysaccharide (LPS) (Heremans et al., J. Exp. Med. 1990. 171: 1853): treatment of mice with neutralizing monoclonal antibody against IFN-gamma was found to completely prevent lethal shock reactions, in particular the generalized Shwartzman reaction, whereas treatment with IFN-gamma sensitized the mice to the development of such reactions. Since activated T cells and natural killer (NK) cells are the main if not the only potential source of LPS-induced IFN-gamma, we investigated the relative importance of these cells in the development of the generalized Shwartzman-like reaction in mice by depleting them selectively with relevant monoclonal antibodies. Treatment with antibodies directed against the CD4+ T cells subset was not effective in protecting mice. Anti-CD8 antibody did attenuate the reaction to some extent. However, markedly reduced mortality was seen in mice which were depleted of NK cells by systemic administration of polyclonal anti-asialo GM1 or monoclonal anti-NK1.1 antibodies. Failure of T cells to promote the Shwartzman reaction was also evidenced by the observation that thymus-less nude mice, which are deficient in T cells, were more rather than less sensitive to the reaction. Approximately 20 times less LPS was needed to induce the lethal reaction in these mice than in NMRI mice and 58 times more anti-IFN-gamma antibody was required to block mortality. Nu/nu mice reportedly have an over-active NK cell compartiment. IFN-gamma production by these cells in LPS-treated mice may account for the augmented sensitivity. Our data suggest that NK cells may be the most important source of endogenous IFN-gamma which mediates the LPS-induced lethal reactions in mice.

Porphyromonas gingivalis 381 lipid A, characterized by beta-(1-6)-linked glucosamine disaccharide 1-phosphate, with one hydroxyacyl group and one acyloxyacl group, i.e., 3-hydroxy-15-methyl-hexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2 and 2' positions, respectively, was less endotoxically active than the synthetic Escherichia-coli-type lipid A (compound 506), which possesses beta-(1-6)-linked glucosamine disaccharide 1,4'-bisphosphate, with two acyloxyacyl groups at the 2' and 3' positions and two 3-hydroxytetradecanoyl group at the 2 and 3 positions and the synthetic Salmonella-type lipid A (compound 516), which has three acyloxyacyl groups at the 2,2' and 3' positions and one hydroxyacyl group at the 3 position. P. gingivalis lipid A exhibited no or very low endotoxic activities, i.e., lethal toxicity in galactosamine-loaded mice, preparative ability for local Shwartzman reaction, pyrogenicity and Limulus test as compared with compounds 506 and 516. However, polyclonal B-cell activation of BALB/c mouse splenocytes was as strong as that of compound 506. Furthermore, P. gingivalis lipid A had stronger immunoadjuvant and hemagglutinating activities than compound 506. The absence of ester-linked phosphate at the 4' position and ester-linked fatty acids, and the presence and positions of fatty acids possessing considerable lengths of acyl chains are unique features of P. gingivalis lipid A, and they differentiate this lipid A from enterobacterial and other lipids A. The good balance between endotoxic properties and beneficial immunobiological activities of P. gingivalis lipid A may be attributable to these features.

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.

Intravenous injection of E. coli culture caused a sudden death in rabbits which were injected Streptococcus culture into the myocardium 24 h before the experiment. Pretreatment of animals with Trasylol-R, before injection of E. coli culture, caused an increase of survival time. In isolated Langendorff perfused heart from myocardially Streptococcus culture injected animals, E. coli culture filtrate caused a transitory increase and then a continuous decrease in coronary perfusion pressure together with an increase in heart rate. These findings are shown to be prevented by previous addition of Trasylol to the perfusion medium. According to these results it is speculated that a Shwartzman-like phenomenon can also occur in the heart muscle. The possible role of the activation of tissue kallikrein system in the production of this phenomenon is discussed.

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.

The local Shwartzman reaction occurred in germfree rabbits which had no natural antibody to endotoxin and none or only a very small amount of immunoglobulin G. From the results it was concluded that the presence of natural antibody to endotoxin is not a prerequisite of the production of the local Shwartzman reaction by bacterial endotoxin.

Pseudomembranous colitis is a potentially fatal disorder associated with gastrointestinal surgery and the use of antibiotics. The aetiological agent has been shown to be Clostridium difficile but the pathogenesis of the disease is unknown. It has been suggested that the lesions produced are due to a local Shwartzman effect. Such an effect may be accompanied by activation of the serum complement system and we therefore looked for evidence of complement consumption in 4 patients with the diagnostic clinical and histological features of pseudomembranous colitis. Positive evidence was obtained suggesting that a Shwartzman phenomenon may be involved.

The hypothesis of acute renal failure which now best fits the facts is that it is a Shwartzman equivalent, due to the multiple noxious effects of endotoxin. Endotoxin alone can account for all those separate factors which are known to cause an increase of the renal vascular resistance. The discrepancies and the implications are discussed.

Fluorescein-labeled antigen-antibody complexes could be made visible by scanning electron microscopy using an intensifying device. This new method of immunocathodoluminescence was demonstrated on cryostat sections of rat kidneys containing glomerular fibrin as the result of endotoxin infusion. The resulting photographs correspond with those obtained by immunofluorescent microscopy. The advantage of this technique is, however, the larger depth of focus. By using thinner cyostat sections it is expected that the higher resolution of scanning microscopy will provide even better details in three dimensions.

The occurrence of the Local Shwartzman Reaction (LSR) in equine species has not previously been reported. The molecular mechanism appears identical to that reported for the rabbit and other species. The immunopathologic and histopathologic similarities of the experimentally induced LSR in horses and ponies to that of the hoof-laminae (an extension of the skin) lesion in naturally-occurring and/or carbohydrate induced laminitis may offer insight into the pathogenesis of this complex disease.

Four albino rabbits received two intradermal injections of endotoxin followed in 24 hours by an intravenous injection of endotoxin. All animals developed the local Shwartzman reaction, characterized by hemorrhagic necrosis over each intradermal injection site. Light microscopy of the Shwartzman lesion showed intense inflammation and vascular thrombosis. Frozen sections of the skin lesion revealed marked vascular deposition of fibrin with lesser amounts of IgG, IgM, C3, and albumin. Light microscopy was normal and immunofluorescence was negative in skin obtained prior to the initial injection of endotoxin and skin adjacent to the Shwartzman lesion. These findings suggest nonspecific trapping of serum proteins within vascular thrombi and indicate that immune mechanisms may not play a role in the pathogenesis of the local Shwartzman reaction.

The ability of antisera to guinea-pig C3 to inhibit the Arthus and local Shwartzman reactions was studied. They were found to reduce the non-haemorrhagic component of the active and reversed passive Arthus reactions and to delay the appearance of the haemorrhage in the active Arthus reaction. Cobra venom factor, however, had no effect on the non-haemorrhagic components of these reactions and only delayed the appearance of the haemorrhage of the active Arthus reaction. There appeared to be a correlation between the serum complement level and the time taken for the haemorrhage to appear, and between the circulating platelet count and the extent of the non-haemorrhagic, oedematous component of the reaction. The haemorrhagic component of the local Shwartzman reaction was not affected by decomplementation with cobra venom factor. The ability of the antisera to inhibit the haemorrhage of the Shwartzman reaction was not dependent on lowering the serum complement titre. However, the haemorrhage was inhibited if the circulating platelet count was also reduced to very low numbers. Antiserum to zymosan alone had the same effect as anti-beta1C/beta1A globulin (zymosan) in blocking the reaction, although it did not alter the complement levels or the platelet counts. The possibility of an immunological cross-reactivity between zymosan and endotoxin in this action is discussed.

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.