Potentiation of lethal endotoxin shock by streptococcal pyrogenic exotoxin in rabbits: Possible relevance of hyperreactivity of macrophages to endotoxin

Abstract Streptococcal pyrogenic exotoxin (SPE) potentiates lethal shock induced by endotoxin. We have previously reported that macrophages derived from SPE-treated rabbits showed hyperreactivity to endotoxin, and that the effect of SPE on macrophages was mediated by a lymphokine(s). Here we show that culture supernatants of SPE-stimulated lymphocytes, when administered into rabbits three hours before or together with endotoxin, potentiate a variety of endotoxin-induced pathophysiological changes and even lethal shock. These results suggest that SPE-induced lymphokine(s) mediates the potentiating effect of SPE on the lethal endotoxin shock through enhancing endotoxin reactivity of macrophages which play the central role in mediating endotoxin toxicity.     

Ganglioside expression in macrophages from endotoxin responder and nonresponder mice

Peritoneal macrophage ganglioside patterns and ganglioside sialic acid content were compared for two congenic strains of mice having differing responses to bacterial lipopolysaccharide. Resident macrophage ganglioside patterns from C3H/HeJ mice (endotoxin hyporesponsive) and C3H/HeN mice (endotoxin responsive) were similar. Macrophages elicited with phenol-extracted or butanol-extracted endotoxin showed distinctly more complex ganglioside patterns in C3H/HeN mice. C3H/HeJ macrophages showed distinct, but less complex changes when elicited with butanol-extracted endotoxin. As expected, there were minimal alterations induced by phenol-extracted endotoxin in the C3H/HeJ patterns. When injected with whole killed E. coli, both strains of mice exhibited complex ganglioside patterns; however, there were relative differences in the quantities of multiple gangliosides. Differences in ganglioside patterns were mirrored in the relative ratios of N-acetyl- to N-glycolylneuraminic acid. When macrophages were activated by administration of either endotoxin preparation, macrophage gangliosides from C3H/HeN mice always contained a higher proportion of N-acetylneuraminic acid compared with C3H/HeJ macrophage gangliosides. Oxidative metabolism of the macrophage populations was assessed by PMA-induced H2O2 release. This indicated that endotoxin activation produced an increase in PMA-induced H2O2 release as well as a shift of sialic acid class from the N-glycolyl type to the N-acetyl type. However, no direct correlation could be made between ganglioside composition, sialic acid content, and macrophage function. These data indicate that both ganglioside composition and sialic acid composition of macrophages are profoundly altered with endotoxin activation. The data further indicate that under conditions which C3H/HeJ mice respond to Gram-negative bacteria, their macrophage ganglioside patterns still differ from normal mice.     

Maternal fever at parturition and its effects on the newborn rabbit.

The febrile response to administration of endotoxin has been reported to be suppressed in both pregnant animals at term and in their newborn. In a previous study we found that newborn rabbits under appropriate conditions to develop a febrile reaction to injected endotoxin. In this investigation we sought to discover whether pregnant rabbits at term had a febrile response to endotoxin, and if so, its effect on thermoregulation in their newborn. Endotoxin (E. Coli LPS) was injected into 19 pregnant rabbits at term. Six delivered spontaneously within an hour. At one hour, 13 were given oxytocin, and a further 8 delivered within five minutes. The colonic temperature (Tc) of the mothers before endotoxin administration and at delivery, and of their young, was measured. The results were compared with those of 10 pregnant rabbits not given endotoxin, and their young. Within 15 min of delivery newborn rabbits from each litter were placed on a thermal gradient to assess their thermoregulatory responses. Pregnant rabbits at term developed an impressive febrile response to injected endotoxin and their young were born with high colonic temperatures. Newborn rabbits from febrile mothers selected higher thermal environments and maintained a higher colonic temperature than the newborn of non-febrile mothers. We conclude that fever is sustained in the first hours of life in the newborn of mothers injected with endotoxin. The possible mechanisms are of considerable interest. None of the pregnant rabbits died after endotoxin administration, but the stillbirth rate was 50% compared with 10% in non-febrile does.     

Cellular immunity induced by avirulent Salmonella in LPS-defective C3H/HeJ mice

An avirulent strain of Salmonella, SL3235, has been shown to confer high levels of immunity on lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice. Immunized mice were also protected against challenge with Listeria monocytogenes, indicating that the Salmonella vaccine activates macrophages. It was shown that protection and macrophage activation occurred without correction of the LPS defect, as assessed by in vivo endotoxin toxicity, in vitro spleen cell mitogenicity, and the ability of in vivo treatment with LPS to enhance in vitro macrophage ingestion of C3b-coated erythrocytes. It is concluded that LPS responsiveness is neither a necessary nor a sufficient condition for Salmonella immunity, and that macrophage activation can apparently occur in C3H/HeJ mice in the face of a sustained LPS defect.     

GRP94/gp96 elicits ERK activation in murine macrophages. A role for endotoxin contamination in NF-kappa B activation and nitric oxide production

Vaccination of mice with GRP94/gp96, the endoplasmic reticulum Hsp90, elicits a variety of immune responses sufficient for tumor rejection and the suppression of metastatic tumor progression. Macrophages are a prominent GRP94/gp96 target, with GRP94/gp96 reported to activate macrophage NF-kappa B signaling and nitric oxide production, as well as the MAP kinase p38, JNK, and ERK signaling cascades. However, recent studies report that heat shock protein elicited macrophage activation is due, in large part, to contaminating endotoxin. To examine the generality of this finding, we have investigated the role of endotoxin in GRP94/gp96-elicited macrophage activation. We report that GRP94/gp96 binds endotoxin in a high-affinity, saturable, and specific manner. Low endotoxin calreticulin and GRP94/gp96 were purified, the latter using a novel method of depyrogenation; this resulted in GRP94/gp96 and calreticulin preparations with endotoxin levels substantially lower than those of previously reported preparations. Low endotoxin GRP94/gp96 retained its native conformation, ligand binding activity, and in vitro chaperone function, yet did not activate macrophage NF-kappa B signaling, nitric oxide production or inducible nitric-oxide synthase production. Low endotoxin GRP94/gp96 and calreticulin did, however, elicit a marked increase in ERK phosphorylation at protein concentrations as low as 2 microg/ml. These results are discussed with respect to current understanding of the contributions of endotoxin and heat shock/chaperone proteins to the stimulation of innate immune responses.     

Age-related differences in thermoregulatory responses to endotoxin in rabbits.

E. coli endotoxin evokes fever in rabbits immediately after birth. In 0--3 day-old rabbits the fever is monophasic and brown fat thermogenesis is mainly responsible for the reaction. In 6--10 day-old animals the fever is usually biphasic and increased heat conservation also contributes to the response. An inverse relationship exists between the endotoxin dose and the latent period before the onset of fever, while the height of the fever is independent of the endotoxin dose. The response is similar as that of adult rabbits, except that after all endotoxin doses the latent period is longer and the magnitude of the response slightly smaller in the newborn.     

Effect of streptococcal pyrogenic exotoxin on rabbit macrophage functions in vitro: mediation by splenic lymphocytes

Streptococcal pyrogenic exotoxin (SPE) showed no direct effect on rabbit macrophage functions in vitro. However, when splenic lymphocytes were added to macrophage cultures, SPE caused marked augmentation of glucose consumption and superoxide anion production, and concomitant inhibition of phagocytosis without loss of cell viability. The SPE effects were demonstrated to be mediated by a soluble factor(s) released from the splenic lymphocytes in response to SPE stimulus.     

Development of a highly sensitive in vitro assay method for biological activity of endotoxin contamination in biological products.

The pyrogen test in rabbits has been replaced by the bacterial endotoxin test. The endotoxin test, however, showed a considerable discrepancy with pyrogenicity and was, therefore, assumed to have an efficacy limitation in directly predicting harmful biological effects of endotoxin. We developed a sensitive in vitro assay method by making use of tumour necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells, which showed a fine correlation with pyrogenicity in rabbits. RAW264.7 cells maintained by serial subculture under an endotoxin-free condition have gained the similar level of sensitivity as the endotoxin test to allow extensive dilutions of a drug for eliminating adverse effects on the cells. The in vitro TNF-alpha induction assay was shown to be capable to detect quantitatively a synergistic effect of a drug and endotoxin. The synergy is assumed necessary to be taken into consideration to define the limit value for the endotoxin test for guaranteeing the similar level of safety as by the pyrogen test.     

Further studies on the activation of lymphocytes by endotoxin protein.

Endotoxin protein, an outer membrane protein of Gram-negative bacteria associated with lipopolysaccharide endotoxin, has been found to be a potent activator of lymphocytes. In the absence of T lymphocytes and macrophages, endotoxin protein can stimulate murine B lymphocytes to synthesize DNA and produce antibodies of diverse specificities. This stimulation is greater than that obtained with two well known murine B cell activators, lipopolysaccharide endotoxin or PPD-tuberculin. Splenic lymphocytes from other species, such as rats, rabbits, and guinea pigs also proliferate when cultured with endotoxin protein. Of particular significance is the finding that endotoxin protein is an activator of human peripheral blood lymphocytes.     

Changes in membrane gangliosides: differentiation of human and murine monocytic cells

Membrane ganglioside changes in murine peritoneal macrophages and the human promyelocytic leukemia cell line HL-60 have been assessed by two-dimensional thin-layer chromatography. C3H/HeJ mice respond to protein-containing endotoxin but are hyporesponsive to protein-free endotoxin preparations. Compared to unstimulated resident cells, protein-containing endotoxin produced an alteration in the C3H/HeJ macrophage ganglioside pattern whereas protein-free endotoxin did not. In comparison, differentiation of HL-60 cells to a neutrophil-like cell by dimethylsulfoxide gave a ganglioside pattern similar to unstimulated HL-60 cells. However, differentiation of HL-60 cells by phorbol myristate acetate to macrophage-like cells results in a large increase in the monosialoganglioside GM3. The evidence presented indicates that discrete ganglioside changes occur in murine monocytes and HL-60 cells upon induction to cells with increased macrophage functions.     

浙江省热原检查用兔敏感性状况调查

目的调查浙江省热原检查用兔对细菌内毒素的敏感性情况,为提高热原检查结果的准确性和可靠性提供参考。方法对全省所有取得生产许可证单位的家兔,用国家颁发的细菌内毒素标准品,＂热原检查法＂进行检查,剂量分别为5EU/Kg和10EU/Kg,记录并比较各单位家兔的平均升温值和升温率。结果对细菌内毒素,各兔场家兔的敏感性有一定的差异。静脉注射5EU/kg,平均升温值为0.40℃～0.87℃,升温率为35%～83%;静脉注射10EU/kg,平均升温值为0.74℃～1.16℃,升温率为70%～94%。结论不同生产单位的家兔对细菌内毒素的敏感性不同,有必要对热原检查用家兔进行细菌内毒素敏感性检查。     

Local Schwartzman phenomenon in axenic rabbits]

Local Schwartzman phenomenon was produced by coli-endotoxin in all the germfree rabbits tested (11 in all) at the age of 102 to 135 days. Any kinds of natural antibodies were not detected in sera of the rabbits, which in fact were found to be agammaglobulinaemic in most cases, as revealed by immunoelectrophoresis. These facts suggested that the germfree rabbits utilized here had not been sensitized to bacterial endotoxins. From the results obtained here, it may be concluded that the existence of hypersensitivity to endotoxin is not necessary to the production of local Schwartzman phenomenon by bacterial endotoxin.     

Bromelain activates murine macrophages and natural killer cells in vitro

The innate immune response is critical for effective immunity against most pathogens. In this study, we show that bromelain, a mixture of cysteine proteases, can enhance IFN-gamma-mediated nitric oxide and TNFalpha production by macrophages. Bromelain's effect was independent of endotoxin receptor activation and was not caused by direct modulation of IFN-gamma receptors. Instead, bromelain either enhanced or acted synergistically with IFN-gamma receptor-mediated signals. These effects were seen in both RAW 264.7, a macrophage cell line, and primary macrophage populations. Bromelain also increased IL-2- and IL-12-mediated IFN-gamma production by NK cells. These results indicate a potential role for bromelain in the activation of inflammatory responses in situations where they may be deficient, such as may occur in immunocompromised individuals.     

Effect of bacterial endotoxin on the phagocytic activity of the reticulo-endothelial system in rabbits of different age

Stimulation of the phagocytic activity of the reticulo-endothelial system with bacterial endotoxin was studied in newborn rabbits in the period in which they do not actively form antibodies to bacterial antigens. A markedly accelerated clearance of colloidal carbon from the blood was found in five-day-old rabbits (tested by the technique of Biozzi, Benacerraf and Halpern 1953) when relatively high doses of endotoxin were used. It is assumed that the increased resistance to infection which may be elicited in young animals in that period is due to stimulation of cellular defence mechanisms. By comparison of the stimulating effect of endotoxin on phagocytosis in five-day-old, one-month-old and adult rabbits it was found that the phagocytic cells of the R.E.S. are more susceptible to the effect of endotoxin in adults than in newborns. This difference is evident from comparisons of the phagocytic indices K and the corrected phagocytic indices α in three age groups of rabbits stimulated with different doses of endotoxin. The possible mechanism and cause of differences in sensitivity of young and adult individuals is discussed.     

In vitro generation of K562 killers in human T-lymphocyte subsets

Group A streptococcal pyrogenic exotoxin (SPE) is a potent modulator of the immune system when used experimentally in mice. Typically, a late burst of plaque-forming cells (PFC) follows an early suppression of the antibody response in appropriately immunized and SPE-treated mice or their spleen cells in vitro. This altered response to antigen caused by SPE is termed a deregulated antibody response. The site of action of SPE was studied by use of cellular reconstruction and complementation experiments using the separated subpopulations of immunocytes which are required for full expression of mouse spleen PFC responses to sheep erythrocytes or to trinitrophenylated (TNP) rabbit erythrocytes in vitro. The SPE site was thus localized to the T-cell subpopulation. Recently SPE has been purified to a very high degree, making it possible to ascertain that SPE alone generates the deregulation of the immune system as described before and to limit the role of nondefined components of cruder preparations of SPE. A purified horse anti-scarlet fever antitoxin which recognizes highly purified SPE as being homogeneous also recognized a single component of crude SPE by agar-gel analysis. A rabbit anti-SPE immunoglobulin raised against crude SPE and absorbed with killed, strain NY5, Group A streptococci recognized the pure SPE and a major component of the homologous crude SPE similarly. Both of these antisera neutralized the capacity of SPE to deregulate the in vitro PFC response to TNP almost completely. A third antiserum raised in rabbits against a NY5 Group A streptococcal whole cell vaccine recognized a different component of crude SPE and totally failed to recognize pure SPE. This antiserum also recognized a purified Group A streptococcal peptidoglycan as being related to components contained in the crude SPE preparation. This antiserum, however, totally failed to neutralize the capacity of SPE to deregulate the PFC response to TNP. These results show that SPE-A is the active component of cruder preparations of SPE which deregulates PFC responses.     

Endotoxin-induced size change in bone marrow progenitors of granulocytes and macrophages

Injection of 5 μg endotoxin to adult C₅₇BL mice caused a marked increase in the sedimentation velocity of granulocytic and macrophage progenitor (colony-forming) cells in the bone marrow. This change was maximal two days after injection and was not accompanied by corresponding changes in total marrow nucleated cell populations. The endotoxin-induced shift was not dependent on the presence of the thymus but did not occur in mice challenged after preinjection with endotoxin. No changes in buoyant density, cell cycle status, pattern of differentiation and responsiveness of granulocytic and macrophage progenitor cells were observed after the injection of endotoxin. The increased sedimentation velocity of progenitor cells appears to indicate an increase in cell volume but the mechanisms involved have not been identified.     

Inhibition of erythroid differentiation of mouse erythroleukemia cells by a macrophage product(s)

Conditioned media from established murine macrophage cell lines (RAW264.7, P388D1, and WEHI-3) incubated with endotoxin in a serum-free medium contain an erythroid inhibitory activity (EIA) that inhibited dimethylsulfoxide-induced erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells. Endotoxin itself has no EIA activity. Partial purification of EIA demonstrated that it is distinct from other macrophage products such as IL-1, TGF beta, ECGF, FGF, G-CSF, hepatocyte stimulating factor, interferon, PDGF, and cachectin/TNF. These findings indicate that EIA is a macrophage product distinct from other monokines.     

Endotoxin tolerance is associated with altered GTP-binding protein function.

Previous studies have suggested that guanine nucleotide regulatory (G) proteins modulate endotoxin-stimulated peritoneal macrophage arachidonic acid (AA) metabolism. Endotoxin-stimulated metabolism of AA by peritoneal macrophages is decreased in endotoxin tolerance (Rogers et al. Prostaglandins 31: 639-650, 1986). These observations led to a study of G protein function and AA metabolism by peritoneal macrophages in endotoxin tolerance. Endotoxin tolerance was induced by the administration of sublethal doses of endotoxin. AA metabolism was assessed by measurement of thromboxane B2 (TxB2), a cyclooxygenase metabolite. NaF (5 mM), an activator of G proteins, significantly stimulated TxB2 synthesis in control macrophages from 7.7 +/- 0.2 to 19.1 +/- 0.6 (SE) ng/ml (P less than 0.05) at 2 h and was partially inhibited by pertussis toxin, suggesting a G protein-dependent mechanism. Salmonella enteritidis endotoxin (50 micrograms/ml) stimulated a similar increase in TxB2 levels (23 +/- 0.4 ng/ml, P less than 0.05). In contrast to control macrophages, macrophages from endotoxin-tolerant rats stimulated with either NaF or S. enteritidis endotoxin had TxB2 levels that were only 30 and 2% of the respective stimulated control cells. Basal guanosine-triphosphatase (GTPase) activity (33 +/- 6 pmol.mg-1.min-1) in endotoxin-tolerant macrophage membranes was significantly lower (P less than 0.05) than control basal activity (158 +/- 5 pmol.mg-1.min-1). This suppression of macrophage GTPase activity was apparent 48 h after the first in vivo sublethal endotoxin injection (100 micrograms/kg ip). The reduced GTPase activity paralleled in vitro cellular hyporesponsiveness to endotoxin-stimulated TxB2 production.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Gram-negative bacterium Chlamydia trachomatis L2 stimulates tumor necrosis factor secretion by innate immune cells independently of its endotoxin

The endotoxin of Chlamydia trachomatis L(2), the causative agent of lymphogranuloma venerum, has been described as an endotoxin with an atypical structure and weak stimulatory activity. It is, however, unclear whether chlamydial endotoxin plays a role in the stimulation of innate immune cells upon contact with the whole microorganism C. trachomatis L(2). We show here that chlamydial endotoxin and, as expected, Escherichia coli O55:B5 endotoxin depend on Toll-like receptor 4 without depending on Toll-like receptor 2 to stimulate bone marrow-derived dendritic cells to secrete tumor necrosis factor (TNF). In contrast, the whole microorganism C. trachomatis L(2) induces TNF secretion by innate immune cells independently of Toll-like receptor 4, while stimulation by E. coli O55:B5 depends on Toll-like receptor 4. Furthermore, although TNF secretion of the macrophage cell line RAW264.7 with chlamydial or E. coli O55:B5 endotoxin as well as with the bacterium E. coli O55:B5 is inhibited by the endotoxin-neutralizing compound polymyxin B, C. trachomatis L(2)-induced secretion of TNF cannot be reduced. In accordance with the literature, the potential of chlamydial endotoxin is more than 100-fold weaker than E. coli O55:B5 endotoxin on all cell types tested. We conclude that chlamydial endotoxin is unlikely to be involved in C. trachomatis L(2)-induced release of TNF by innate immune cells.