Peritoneal Lavage Reduces Lipopolysaccharide-Induced Elevation of Serum TNF-α and IL-6 and Mortality in Mice

The contribution of peritoneal cells to lipopolysaccharide (LPS)-induced elevation of serum TNF- and IL-6 levels and mortality has been studied. Peritoneal lavage performed before LPS administration reduced serum cytokine levels by approximately 50% and mortality from 50 to 100%. The effect of peritoneal lavage is due to the removal of peritoneal cells as reinjection of peritoneal cells eliminated the protective effect of lavage on LPS-induced mortality. A special role of peritoneal macrophages in the systemic response to LPS was suggested by the finding that LPS-induced an increase in intracellular TNF- and IL-6 in peritoneal macrophages but in neither splenic nor bone marrow macrophages. Intraperitoneal injection of thioglycollate broth 4 days prior to lavage increased the number of peritoneal cells removed by lavage and increased protection from LPS mortality. Peritoneal lavage performed 30 to 120 minutes after the LPS administration completely protected all mice from LPS-induced mortality, suggesting the possibility that such treatment may offer a novel therapeutic approach to septic shock.     

The role of macrophages in LPS-induced lethality and tissue injury.

In the present study we investigated the role of mononuclear phagocytes in the pathogenesis of lipopolysaccharide (LPS)-induced lethality and tissue injury. Since hepatic and splenic macrophages are the primary sites of localization of i.v.-injected LPS, we selectively eliminated these macrophages using liposome-encapsulated dichloromethylene diphosphonate (DMDP). After double DMDP-liposome treatment the phagocytic cells in the liver and spleen were completely eliminated, except for the macrophages in the white pulp of the spleen which were affected to a lesser extent by this treatment. An i.v. injection of LPS into DMDP- and saline-pretreated mice showed that the latter animals exhibited febrile-associated symptoms such as lethargy and ruffled fur, but that macrophage elimination abrogated these symptoms. Although after double saline- or DMDP-pretreatment the LD50 appears to be 1 mg and 630 micrograms, respectively, the differences in lethality between both groups of mice were not statistically significant. Therefore, we concluded that hepatic and splenic macrophages are not necessary for LPS-induced lethality. The role of macrophages in LPS-induced local tissue damage was studied by comparing the histopathological changes in hepatic and splenic tissue between DMDP- and saline-pretreated mice. A sublethal dose of LPS induced similar hepatic lesions in macrophage-depleted and saline-pretreated mice, whereas the histopathological changes in the spleen were much more pronounced after DMDP-pretreatment. Particularly in the inner periarteriolar lymphocyte sheath (PALS) of these mice, the number of T cells was considerably reduced and extensive cellular necrosis could be found. These data strongly suggest that the local tissue damage resulting from LPS injection may not be due to its localization in mononuclear phagocytes but rather to interaction with other cell types.     

Strain-dependent cytotoxic effects of endotoxin for mouse peritoneal macrophages.

The cytotoxic effects of bacterial lipopolysaccharides (LPS) on mouse leukocytes have been examined in vivo and in vitro. Intraperitoneal injection of LPS into C57BL/6 mice greatly reduced the recovery of mononuclear cells; LPS was cytotoxic for macrophages, but had a mitogenic effect on lymphocytes. Similar effects of LPS on peritoneal leukocytes were observed in vitro. When monolayers of adherent peritoneal cells were studied in vitro, cytotoxicity was also observed, suggesting that the effect of LPS on macrophages is direct and does not require participation by lymphocytes. Entirely different results were obtained when peritoneal macrophages from LPS-resistant C3H/HeJ mice were studied. LPS failed to activate lymphocytes and was not cytotoxic for macrophages in vitro or in vivo. The effect of LPS on polymorphonuclear leukocytes appeared to be the same in all mouse stains studied. Lipid A was shown to be the most biologically active portion of the LPS molecule. Whereas polysaccharide-deficient endotoxins extracted from rough mutants of Salmonella typhimurium were cytotoxic for macrophages in vitro, polysaccharides that lacked esterified fatty acids did not exhibit this activity. Since LPS may mediate its effects through affinity for mammalian cell membranes, the cellular unresponsiveness of C3H/H3J mice to LPS may reflect an inability of cells from LPS-resistant strains to interact with LPS at the membrane level.     

Thymulin, a thymic peptide, prevents the overproduction of pro-inflammatory cytokines and heat shock protein Hsp70 in inflammation-bearing mice

The effects of synthetic analogue of peptide hormone thymulin, which is normally produced by thymic epithelial cells, on immune cells activity and blood cytokine profile had been studied in male NMRI mice with acute inflammation induced by injection of lipopolysaccharide from gram-negative bacteria (LPS, 250 microg/100 g of body weight). Inflammation induced by LPS resulted in accumulation of several plasma pro-inflammatory cytokines, IL-1 beta, IL-2, IL-6, TNF-alpha, interferon-gamma, and also IL-10, anti-inflammatory cytokine. Thymulin previously injected in dose of 15 microg/100 g body weight, prevented the accumulation of proinflammatory cytokines in plasma. Thymulin also prevented LPS-induced up-regulation of production of several cytokines by spleen lymphocytes and peritoneal macrophages. Added in vitro, thymulin decreased the peak of TNF-alpha production in macrophages cultivated with LPS. In addition, thymulin lowered the peak of Hsp70 production induced by LPS treatment. The results indicate that thymulin having significant anti-inflammatory effect may be promising in clinical application.     

Effects of bilirubin on murine peritoneal and spleen cells

The effect of bilirubin on murine peritoneal and spleen cells was investigated. Bilirubin was found to have a strong and rapid effect on the expression of various kinds of Fc receptors on peritoneal macrophages. Significant changes were observed 30 min after the infection of bilirubin. The return to normal values was not observed earlier than after 24 h. The effect of bilirubin on Fc receptor expression of splenic macrophages was less pronounced. Expression of Ia antigen on macrophages was not influenced by bilirubin. The changes in percentage of sIg+ and Thy 1.2+ lymphocytes reflect a change in the ratio of T to B cells in the peritoneal cavity, as bilirubin caused 40% increase in numbers of B cells and a similar decrease in numbers of T cells. The percentage of splenic B lymphocytes was not influenced by bilirubin injection; but the ratio of T helper to suppressor cells was altered.     

Modulation of macrophage Ia expression by lipopolysaccharide: stem cell requirements, accessory lymphocyte involvement, and IA-inducing factor production.

The mechanism of induction of murine macrophage Ia expression by lipopolysaccharide (LPS) was studied. Intraperitoneal injection of 1 microgram of LPS resulted in a 3- to 10-fold increase in the number of IA-positive peritoneal macrophages (flow cytometry and immunofluorescence and a 6-to 16-fold increase by radioimmunoassay. The isolated lipid A moiety of LPS was a potent inducer of macrophage Ia expression. Ia induction required a functional myelopoietic system as indicated by the finding that the response to LPS was eliminated in irradiated (900 rads) mice and reinstated by reconstitution with bone marrow cells. Comparison of LPS-induced Ia expression in normal and LPS-primed mice revealed a faster secondary response to LPS. The memory response could be adoptively transferred to normal mice with nonadherent spleen cells prepared 60 days after LPS injection. Spleen cells prepared 5 days after LPS injection caused Ia induction in LPS-nonresponder mice; such induction was not observed in irradiated (900 rads) recipients. The cell responsible for this phenomenon was identified as a Thy-1+, immunoglobulin-negative nonadherent cell. The biosynthesis and expression of Ia were not increased by direct exposure of macrophages to LPS in vitro. Small amounts of LPS inhibited Ia induction by gamma interferon. LPS showed positive regulatory effects on Ia expression by delaying the loss of Ia expression on cultured macrophages and by stimulating the production of Ia-inducing factors. Supernatants from cultured spleen cells stimulated with LPS in vitro contained antiviral and Ia-inducing activity that was acid labile, indicating that the active factor is gamma interferon. We conclude that induction of Ia expression by LPS in vivo is a bone-marrow-dependent, radiation-sensitive process which involves the stimulation of a gamma interferon-producing accessory lymphocyte and a delay in Ia turnover.     

Thymulin,A Thymic Peptide,Prevents the Overproduction of Pro-Inflammatory Cytokines and Heat Shock Protein Hsp70 in Inflammation-Bearing Mice

The effects of synthetic analogue of peptide hormone thymulin, which is normally produced by thymic epithelial cells, on immune cells activity and blood cytokine profile had been studied in male NMRI mice with acute inflammation induced by injection of lipopolysaccharide from gram-negative bacteria (LPS, 250 μg/100 g of body weight). Inflammation induced by LPS resulted in accumulation of several plasma pro-inflammatory cytokines, IL-1β, IL-2, IL-6, TNF-α, interferon-γ, and also IL-10, anti-inflammatory cytokine. Thymulin previously injected in dose of 15 μg/100 g body weight, prevented the accumulation of proinflammatory cytokines in plasma. Thymulin also prevented LPS-induced up-regulation of production of several cytokines by spleen lymphocytes and peritoneal macrophages. Added in vitro, thymulin decreased the peak of TNF-α production in macrophages cultivated with LPS. In addition, thymulin lowered the peak of Hsp70 production induced by LPS treatment. The results indicate that thymulin having significant anti-inflammatory effect may be promising in clinical application.     

Peroxisome proliferator activated receptor gamma is not necessary for the development of LPS-induced tolerance in macrophages

Peroxisome proliferator activated receptor-gamma (PPARgamma) has been reported to exert anti-inflammatory properties in endotoxic shock and sepsis. One phenomenon that alters the inflammatory response to endotoxin [lipopolysaccharide (LPS)] is endotoxin tolerance, which is caused by previous exposure to endotoxin. Here, we investigate whether changes in endogenous PPARgamma function regulate this phenomenon using three different models of LPS-induced tolerance in macrophages. In a first in vitro model, previous LPS exposure of murine J774.2 macrophages suppressed tumour necrosis factor-alpha (TNF-alpha) release in response to subsequent LPS challenge. Treatment of J774.2 cells with the PPARgamma inhibitor GW9662 did not alter tolerance induction because these cells were still hyporesponsive to the secondary LPS challenge. In a second ex vivo model, primary rat peritoneal macrophages from LPS-primed rats exhibited suppression of thromboxane B2 and TNF-alpha production, while maintaining nitrite production in response to in vitro LPS challenge. Pretreatment of rats with the PPARgamma inhibitor GW9662 in vivo failed to alter the tolerant phenotype of these primary macrophages. In a third ex vivo model, primary peritoneal macrophages with conditional deletion of PPARgamma were harvested from LPS-primed Cre-lox mice (Cre+/+ PPARgamma-/-) and exhibited significant suppression of TNF-alpha production in response to in vitro LPS challenge. Furthermore, both LPS-primed PPARgamma-deficient Cre+/+ PPARgamma-/- mice and wild-type Cre-/- PPARgamma+/+ mice exhibited reduced plasma TNF-alpha levels in response to a high dose of LPS in vivo. These data demonstrate that PPARgamma does not play a role in the LPS-induced tolerant phenotype in macrophages.     

In Vivo leukocyte changes induced by Escherichia coli subtilase cytotoxin

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB(5) cytotoxins produced by Shiga-toxigenic Escherichia coli. Its cytotoxicity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum chaperone BiP. Previous studies have shown that intraperitoneal injection of mice with purified SubAB causes a pathology that overlaps with that seen in human cases of hemolytic-uremic syndrome, as well as dramatic splenic atrophy, suggesting that leukocytes are targeted. Here we investigated SubAB-induced leukocyte changes in the peritoneal cavity, blood, and spleen. After intraperitoneal injection, SubAB bound peritoneal leukocytes (including T and B lymphocytes, neutrophils, and macrophages). SubAB elicited marked leukocytosis, which peaked at 24 h, and increased neutrophil activation in the blood and peritoneal cavity. It also induced a marked redistribution of leukocytes among the three compartments: increases in leukocyte subpopulations in the blood and peritoneal cavity coincided with a significant decline in splenic cells. SubAB treatment also elicited significant increases in the apoptosis rates of CD4(+) T cells, B lymphocytes, and macrophages. These findings indicate that apart from direct cytotoxic effects, SubAB interacts with cellular components of both the innate and the adaptive arm of the immune system, with potential consequences for disease pathogenesis.     

Splenic B cells function as immunogenic antigen-presenting cells for the induction of effector T cells

Previous studies performed in our laboratory have revealed that an ordered, sequential, tricellular interaction is obligatory for the antigen-driven induction of a specific effector memory T cell. Thus, it was found that antigen-pulsed peritoneal macrophages signal, in spleen cells, the generation of antigen-specific initiator lymphocytes. These lymphocytes, following injection to syngeneic recipients, recruit, in the draining lymph nodes, "virgin" antigen-reactive T lymphocytes. Although the nature of the first and last cell in the interacting sequence was well characterized, the identity of the intermediary initiator splenic cell was obscure. Studies were carried out to characterize the nature of the splenic initiator cells. It was found that spleen cells from nu/nu, adult thymectomized and neonatal thymectomized, or spleen cells from normal donors which had been subjected to cytolysis using anti-Thy-1.2 antibodies in the presence of complement, did generate, following interaction with keyhole limpet hemocyanin (KLH)-fed macrophages, specific initiator cells. Carrageenan impairment of spleen macrophages did not affect the generation of initiator cells, nor did the depletion of dendritic cells from the spleen. On the other hand highly enriched B cell, but not highly enriched T cell populations, when seeded on KLH-pulsed macrophages, generated antigen-specific initiators, which, in vivo, recruited antigen-reactive T cells. It thus appeared that B lymphocytes can function as intermediary obligatory antigen-presenting cells and actively transfer immunogenic signals from peritoneal antigen-presenting cells to T lymphocytes. These findings may therefore suggest that antigen-specific B cells do not function solely as antibody-producing cells, but, once activated by macrophages, may control the induction and differentiation of some antigen-reactive T cell subsets. Thus, one can view the B cell as an important regulatory cell of both cellular and humoral immune functions. The significance of this observation with regard to Ir gene control at the level of B lymphocytes is discussed.     

Macrophages Suppress Direct B-Cell Activation by Lipopolysaccharide

The influence of macrophages on polyclonal B-cell responses to lipopolysaccharide (LPS) and on a primary, specific immune response to a hapten-LPS conjugate was studied in mouse spleen and lymph node cells in serum-free and serum-supplemented cultures. Macrophages were found not to be necessary, and B cells were directly activated by LPS. In serum-free cultures of macrophage depleted spleen cells, (a) proliferation and antibody secretion occurred at normal levels or were enhanced when compared with normal spleen cells, (b) the responsiveness of limiting cell numbers to LPS was better than in normal spleen cell cultures, (c) LPS did not increase the number or activate residual macrophages, (d) the primary specific response to a hapten-LPS conjugate developed normally, (e) peripheral lymph node cells, which are known to contain a very low concentration of macrophages, from normal or congenitally athymic (nude) mice mounted excellent proliferative responses to LPS Furthermore, in cultures supplemented with fetal bovine serum, depletion of adherent cells resulted in enhancement of LPS-induced B-cell responses. Addition of peritoneal macrophages to adherent cell-depleted spleen cells produced suppression at all concentrations of both mitogen and adherent cells. Suppressive activity could also be demonstrated in supematants from adherent cell cultures stimulated by LPS     

Induction of suppressor macrophages in mice by Fusarenon-X

Intraperitoneal injection of Fusarenon-X into BALB/c mice, a mycotoxin produced by Fusarium nivale Fn 2B, depressed polyclonal antibody formation of splenic lymphocytes in response to pokeweed mitogen (PWM). This inhibitory activity was found to reside in the surface immunoglobulin-negative spleen cell fraction of Fusarenon-X-treated mice, sIg-(FX), which comprised mainly T lymphocytes and smaller number of non-lymphocytic cellular elements. However, reconstitution experiments for in vitro antibody formation provided evidence that T lymphocytes from Fusarenon-X-treated mice, T(FX), which were separated from non-lymphocytic cells by use of carbonyl iron/magnet, were as effective as T lymphocytes from normal mice, T(N), in supporting antibody formation. Furthermore, addition of non-lymphocytic cells, NL(FX), or adherent cells, AD(FX), prepared from spleen cells of Fusarenon-X-treated mice to normal spleen cells strongly inhibited in vitro antibody formation against PWM or a bacterial lipopolysaccharide (LPS). These results strongly indicated that Fusarenon-X induced non-lymphocytic suppressor cells in the spleen of the treated mice which had features in common with activated macrophages.     

S-O_2-1菌苗对小鼠淋巴细胞的作用

动物实验和初步临床应用表明有抑制肿瘤生长作用的S-O_2-1菌苗,在体外能直接刺激小鼠脾细胞淋巴细胞的增殖,并协同增强脾细胞的Con A增殖反应、S-O_2-1菌苗诱导和增强小鼠腹腔粘附细胞的抑制活性,后者表现在直接抑制脾细胞和肠系膜淋巴结细胞对Con A和S-O_2-1菌苗的增殖反应。腹腔转移菌苗活化的腹腔粘附细胞能抑制受体小鼠对SRBC的PFC应答。提示:S-O_2-1菌苗诱导增强巨噬细胞的抑制活性,以致间接抑制淋巴细胞的免疫应答。     

Population kinetics of peritoneal LPS-reactive B lymphocytes

The dynamic behaviour of isolated populations of peritoneal LPS-reactive (LPSr) B lymphocytes was studied upon transfer of peritoneal cells (PerC) from C57BL/6 LPS responder into C57BL/10ScCr LPS non-responder mice. We have followed the persistence and life-span of the transferred LPSr donor B cells in the spleen and peritoneal cavity of both intact and X-irradiated adult hosts after i.v. or i.p injection and neonatal 1-day-old recipients after i.v. transfer. We have found that lymphocyte life-spans can be influenced by local host environments, as the transferred PerC LPSr cells showed different kinetics according to their route of injection, organ localization, and age or state of the recipients. Thus, while in intact hosts most of the transferred peritoneal LPSr cells decayed with time, following transfer into X-irradiated recipients the same cells were able to expand and replenish the lymphoid tissues of the host. Moreover, upon transfer into intact hosts, the kinetic properties of peritoneal LPSr cells from adult mice differ from splenic LPSr cells of age-matched animals, but mimic those of spleen cells from young, 1-to 2-week-old donors. These findings may reflect the different phenotype composition of adult spleen cells (poor in Ly1 B cells) and peritoneal and neonatal spleen cells (both rich for Ly1 B cells), or may be the result of selective events leading to the peritoneal accumulation of cells with different population dynamics.     

In vivo production of heat shock protein in mouse peritoneal macrophages by administration of lipopolysaccharide.

The in vivo production of heat shock protein was studied by administration of bacterial lipopolysaccharide (LPS) into mice. Heat shock protein 70 was detected in the extract of adherent peritoneal cells from mice injected intraperitoneally with LPS by using the immunoblotting method. The expression of heat shock protein 70 was found 2 days after injection of LPS and reached its peak 4 days after injection. The intraperitoneal injection of LPS induced the expression of heat shock protein 70, whereas its subcutaneous injection did not. The in vivo production of heat shock protein 70 was inhibited by administration of LPS together with quercetin, an inhibitor of accumulation of heat shock protein 70 mRNA. Tumor necrosis factor alpha enhanced LPS-induced heat shock protein production in vivo. There was a decrease of gamma delta T cells in the peritoneal cavity of mice injected intraperitoneally with LPS. It was suggested that bacterial LPS is a stressful agent which induces the in vivo heat shock protein response, and its administration leads to the production of heat shock protein 70 in peritoneal macrophages.     

Role of pulmonary macrophages in resistance to experimental metastasis

Intranasal inoculation of C3H/HeN mice with Propionibacterium acnes activates pulmonary macrophages but not splenic or peritoneal macrophages. When mice so treated were injected IV with tumor cells, no protection against the challenge was seen. Conversely, inoculation of C3H/HeN mice with P. acnes by the IP route activated splenic and peritoneal macrophages but not pulmonary macrophages. When mice with activated splenic and peritoneal macrophages were challenged with an IV injection of tumor cells, the mice demonstrated an increased resistance to the formation of pulmonary tumors. Thus, activation of splenic and peritoneal macrophages correlated better with protection against IV tumor challenge than activation of pulmonary macrophages. Experiments were done that demonstrated that the effect was not due to the augmentation of NK cell activity. The data are consistent with the conclusion that activated pulmonary macrophages alone are not effective in conferring resistance to pulmonary tumor nodule formation in this tumor model.     

Effect of a traditional Chinese herbal medicine ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to) on hematopoietic stem cells in mice

Cell dynamics after intraperitoneal (i.p.) and oral administration of a traditional herbal medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to, NYT), were investigated. When NYT was injected i.p. into C3H/He mice, numbers of spleen and peritoneal cells significantly increased in a dose-dependent manner and showed high levels from 4 to 21 days. Two peaks in the total cell number were observed on days 7 and 14 in the peritoneal cavities and spleen of C3H/He mice administered NYT. A marked accumulation of PMN cells in the peritoneal cavity and spleen was detected at 7 days after injection. The numbers of macrophages and lymphocytes also increased by i.p. administration of NYT. The thymus cell number decreased transiently between 4 and 7 days and thereafter returned to the control level. No significant change in the cell number of lymph nodes was observed. Such cellular accumulation was also detected in C3H/HeJ mice, a nonresponder strain to bacterial endotoxin, and athymic nude mice. The activity of colony-forming units in the spleen (CFU-S) of C3H/He as well as C3H/HeJ mice was markedly augmented by i.p. administration of NYT. NYT induced significant CSF production as detectable by its activity in the sera. In addition, oral administration of NYT for 10 days induced a significant increment of peripheral leukocytes and spleen cells and enhanced CFU-S activity of bone marrow cells as induced by i.p. administration, indicating that NYT acts on hematopoietic stem cells capable of differentiating to lymphocytes, macrophages and PMN cells into the periphery.     

Resistance of C58 mice to primary systemic herpes simplex virus infection: macrophage dependence and T-cell independence.

The relative contribution of thymus-derived lymphocytes (T-cells) and of macrophages to resistance to primary infection with herpes simplex virus type 1 (HSV-1) was studied in C58 mice. Resistance was dependent on macrophage competence, but was relatively independent of T-lymphocyte competence. Although aging mice became progressively more deficient in functional T-cells, as demonstrated by a decreasing resistance to transplanted line Ib leukemia and by declining responses to T-cell nitogens (concanavalin A and phytohemagglutinin), their resistance to HSV-1 increased with increasing age. Moreover, in mice that were made selectively deficient in T-cells by the combination of adult thymectomy and treatment with anti-thymocyte serum, resistance to HSV-1 did not correlate spleen and mesenteric lymph nodes. However, selective reduction of macrophages by intraperitoneal injection of silica resulted in enhanced susceptibility to HSV-1. Furthermore, in vitro suppression of HSV-1 plaque formation in mouse embryo fibroblast cells was obtained by cocultivation of infected fibroblast monolayers with peritoneal macrophages, but not with splenic lymphocytes, from adult mice. Macrophages from weanling mice failed to suppress the development of plaques, indicating that the increase in resistance to HSV-1 with age is a result of increased macrophage competence.     

Effects of acute administration of O,O,S-trimethyl phosphorothioate on the respiratory burst and phagocytic activity of splenic and peritoneal leukocytes

An impurity in malathion, O,O,S-trimethyl phosphorothioate (OOS-TMP), was previously shown to be immunosuppressive. The immune cell type which induced immune suppression caused by OOS-TMP at 24 hrs after administration was found to be splenic macrophages. Further characterization of macrophages from OOS-TMP treated mice indicated that OOS-TMP led to macrophage differentiation. In this study, these initial studies were continued and extended to examine the effects of OOS-TMP on splenic and peritoneal macrophages at various times following exposure. Administration of OOS-TMP increased the size heterogeneity of cell volume, phagocytic capability and respiratory burst activity of splenic and peritoneal macrophages. However, by day 7 splenic and peritoneal macrophages from similar to control. These data would suggest that macrophages not previously exposed to OOS-TMP migrated to the spleen and peritoneum of treated animals. This migration may allow the restoration of the ability of splenocytes from treated animals to generate an immune response. Alternatively, these data may indicate that 7 days following exposure to OOS-TMP, the differentiative state of the splenic and peritoneal macrophages of treated mice had decayed and hence these cells had regained resident characteristics.This work was supported by PHS Grant ES04337.