Injection Of Muramyl Dipeptide Research Articles

Postbiotics engage IRF4 in adipocytes to promote sex-dependent changes in blood glucose during obesity.

Postbiotics are microbial‐derived components or metabolites that can influence host immunity and metabolism. Some postbiotics can improve blood glucose control and lower inflammation during bacterial or nutritional stress. Bacterial cell wall‐derived muramyl dipeptide (MDP) is a potent insulin‐sensitizing postbiotic that engages NOD2, RIPK2, and requires interferon regulatory factor 4 (IRF4) to lower inflammation and improve blood glucose. However, the sex‐dependent effects of this postbiotic and the cell type required for IRF4 to cause inflammatory versus glycemic responses to MDP were unknown. Here, we measured how MDP injection altered glucose tolerance and adipose tissue inflammation during low‐level endotoxemia and high fat diet (HFD)‐induced obesity in male and female adipocyte‐specific IRF4 knockout mice (AdipoIRF4fl/fl) compared to WTfl/fl mice. Adipocyte IRF4 was required for the blood glucose‐lowering effects of MDP during endotoxemia and HFD‐induced obesity in male mice. However, MDP did not alter blood glucose in female WTfl/fl and AdipoIRF4fl/f mice during endotoxemia. Unexpectedly, female HFD‐fed AdipoIRF4fl/f mice had lower blood glucose after MDP treatment compared to WTfl/fl mice. MDP lowered inflammatory gene expression in adipose tissue of HFD‐fed WTfl/fl and AdipoIRF4fl/fl mice of both sexes. Therefore, MDP‐mediated lowering of adipose inflammation does not require adipocyte IRF4 and was independent of sex. Together, these data show that injection of MDP, an insulin‐sensitizing postbiotic, lowers adipose tissue inflammation in male and female mice, but lower adipose inflammation is not always associated with improved blood glucose. The blood glucose‐lowering effect of the postbiotic MDP and dependence on adipocyte IRF4 is sex‐dependent.

The Role of the Bacterial Muramyl Dipeptide in the Regulation of GLP-1 and Glycemia.

The host’s intestinal microbiota contributes to endocrine and metabolic responses, but a dysbiosis in this environment can lead to obesity and insulin resistance. Recent work has demonstrated a role for microbial metabolites in the regulation of gut hormones, including the metabolic hormone, glucagon-like peptide-1 (GLP-1). Muramyl dipeptide (MDP) is a bacterial cell wall component which has been shown to improve insulin sensitivity and glucose tolerance in diet-induced obese mice by acting through the nucleotide oligomerization domain 2 (NOD2) receptor. The purpose of this study was to understand the effects of MDP on GLP-1 secretion and glucose regulation. We hypothesized that MDP enhances glucose tolerance by inducing intestinal GLP-1 secretion through NOD2 activation. First, we observed a significant increase in GLP-1 secretion when murine and human L-cells were treated with a fatty acid MDP derivative (L18-MDP). Importantly, we demonstrated the expression of the NOD2 receptor in mouse intestine and in L-cells. In mice, two intraperitoneal injections of MDP (5 mg/kg body weight) caused a significant increase in fasting total GLP-1 in chow-fed mice, however this did not lead to an improvement in oral glucose tolerance. When mice were exposed to a high-fat diet, they eventually lost this MDP-induced GLP-1 release. Finally, we demonstrated in L-cells that hyperglycemic conditions reduce the mRNA expression of NOD2 and GLP-1. Together these findings suggest MDP may play a role in enhancing GLP-1 during normal glycemic conditions but loses its ability to do so in hyperglycemia.

2301-PUB: The Role of Muramyl Dipeptide in Glucagon-Like Peptide-1 Regulation

The host’s intestinal microbiota contributes to endocrine and metabolic responses, but a dysbiosis in this environment can lead to obesity and insulin resistance. A new area a research is focused on understanding how bacterial metabolites contribute to intestinal glucagon-like peptide-1 (GLP-1) release. Muramyl dipeptide (MDP) is a bacterial cell wall component which has been shown to improve insulin sensitivity and glucose tolerance in diet-induced obese mice. The purpose of this study was to understand MDP’s mechanism of action in glucose regulation. We hypothesized that MDP enhances glucose tolerance by inducing intestinal GLP-1 secretion. We observed a significant increase (P<0.001, n=7) in GLP-1 secretion when mouse L-cells were treated with an MDP derivative at 10ug/ml. Using fluorescent immunohistochemistry and RT-PCR we located MDP’s receptor, nucleotide oligomerizing domain 2 (NOD2), in mouse intestine and mouse L-cells. In mice, two intraperitoneal injections of MDP (5mg/kg body weight) or PBS caused a significant increase (P=0.0009, n=15-19) in fasting total GLP-1. Understanding how bacterial products influence GLP-1 secretion and subsequent insulin release, could be translated into novel treatments options for improving glucose regulation in metabolic disease. Disclosure L. Williams: None. A. Alshehri: None. S. Mustafa: None. J. Gagnon: None. Funding Natural Sciences and Engineering Research Council of Canada (2016-05905)

Muramyl dipeptide promotes Aβ1-42 oligomer production via the NOD2/p-p38 MAPK/BACE1 signaling pathway in the SH-SY5Y cells.

The relationship between chronic bacterial colonization in the brain and Alzheimer's disease is attracting extensive attention. Recent studies indicated that the components of bacterial biofilm drive the amyloid-β production. Muramyl dipeptide, the minimal bioactive peptidoglycan motif common to all bacteria, contributes to the development of many central inflammatory and neurodegenerative disorders. However, the involvement of Muramyl dipeptide in amyloid-β production is not completely defined. In our present study, wild type mice received an intracerebroventricular injection of normal saline or Muramyl dipeptide. Data showed that the production of Aβ1-42 oligomers was significantly increased after Muramyl dipeptide injection in the wild type mice or incubation of the SH-SY5Y cells with Muramyl dipeptide. Moreover, the action of Muramyl dipeptide was dose- and time-dependent. The above results suggested a possibility that the Muramyl dipeptide-induced Aβ1-42 oligomer production might be related to the NOD2/p-p38 MAPK/BACE1 pathway. To confirm this, the SH-SY5Y cells were transfected with siRNA NOD2. Data showed that the transfected SH-SY5Y cells exhibited decreased expression of Aβ1-42 oligomer, NOD2, p-p38 MAPK, and BACE1 after treatment with Muramyl dipeptide. Finally, SH-SY5Y cells were pretreated with SB203580, an inhibitor of the p-38-MAPK pathway. The results indicated that these pretreated SH-SY5Y cells exhibited decreased expression of Aβ1-42 oligomer, p-p38 MAPK, and BACE1 after treatment with Muramyl dipeptide. In conclusion, these results suggested that Muramyl dipeptide was the trigger factor for Aβ1-42 oligomer production, which probably acts via the NOD2/p-p38 MAPK/BACE1 signaling pathway.

Role of Muramyl Dipeptide in Lipopolysaccharide-Mediated Biological Activity and Osteoclast Activity.

Lipopolysaccharide (LPS) is an endotoxin and bacterial cell wall component that is capable of inducing inflammation and immunological activity. Muramyl dipeptide (MDP), the minimal essential structural unit responsible for the immunological activity of peptidoglycans, is another inflammation-inducing molecule that is ubiquitously expressed by bacteria. Several studies have shown that inflammation-related biological activities were synergistically induced by interactions between LPS and MDP. MDP synergistically enhances production of proinflammatory cytokines that are induced by LPS exposure. Injection of MDP induces lethal shock in mice challenged with LPS. LPS also induces osteoclast formation and pathological bone resorption; MDP enhances LPS induction of both processes. Furthermore, MDP enhances the LPS-induced receptor activator of NF-κB ligand (RANKL) expression and toll-like receptor 4 (TLR4) expression both in vivo and in vitro. Additionally, MDP enhances LPS-induced mitogen-activated protein kinase (MAPK) signaling in stromal cells. Taken together, these findings suggest that MDP plays an important role in LPS-induced biological activities. This review discusses the role of MDP in LPS-mediated biological activities, primarily in relation to osteoclastogenesis.

Relationship between inflammatory responses and autophagy in lung tissues after scald and NOD2 signaling pathway in septic rats

Objective To evaluate the relationship between inflammatory responses and autophagy in lung tissues after scald and nucleotide-binding oligomerization domain-containing protein 2(NOD2)signaling pathway in septic rats. Methods Twenty SPF healthy male Sprague-Dawley rats, weighing 200-250 g, were divided into control group(group C, n=10) and sepsis after scald group(group SS, n=10)using a random number table.The rats were subjected to a third-degree scald burn covering 20% of total body surface area(body surface was shaved and then exposed to 99-100 ℃ water for 12 s), and 24 h later muramyldipeptide 5 mg/kg was intravenously injected to induce sepsis.The rats were only exposed to 20 ℃ water, and 24 h later normal saline 1 ml was given instead in group C. At 6 h after muramyldipeptide injection in group SS and at 6 h after normal saline injection in group C, arterial blood samples were collected for determination of serum tumor necrosis factor-α and interleukin-6 concentrations by enzyme-linked immunosorbent assay.Then rats were sacrificed and lungs were removed for measurement of activity of myeloperoxidase, NOD2 mRNA expression(using real-time polymerase chain reaction)and expression of receptor interacting protein 2, nuclear factor kappa Bp65 and microtubule-associated protein 1 light chain 3Ⅰ(LC3Ⅰ)and LC3Ⅱ in lung tissues(by Western blot). The LC3Ⅱ/Ⅰratio was calculated. Results Compared with group C, the expression of NOD2 mRNA, receptor interacting protein 2 and nuclear factor kappa Bp65 was significantly up-regulated, and the LC3Ⅱ/Ⅰratio and serum tumor necrosis factor-α and interleukin-6 concentrations were increased in group SS(P<0.05). Conclusion The mechanism underlying enhanced inflammatory responses and autophagy in lung tissues during sepsis after scald may be related to activation of NOD2 signaling pathway in rats. Key words: Burns; Sepsis; Nod2 signaling adaptor protein; Autophagy; Inflammation; Lung

Aberrant interleukin-1 signalling does not increase susceptibility of mice to NOD2-dependent uveitis.

NOD2 is the genetic cause of Blau syndrome, an autoinflammatory disease that manifests as coincident uveitis and arthritis. Since dysregulation of IL-1 signalling is considered a pathogenic mechanism in a number of related autoinflammatory conditions, we examined the extent to which unimpeded interleukin (IL)-1 signalling influences NOD2-dependent inflammation of the eye versus the joint. Mice deficient for IL-1R antagonist (IL-1Ra) were administered the NOD2 agonist muramyl dipeptide (MDP) by systemic (intraperitoneal) or local (intraocular and/or intra-articular) injections. NOD2-deficient mice received an intraocular injection of recombinant IL-1β. Uveitis was evaluated by intravital videomicroscopy and histopathology, and arthritis was assessed by near-infrared imaging and histopathology. Ocular levels of IL-1α, IL-1β and IL-1Ra were quantified by enzyme-linked immunosorbent assay. IL-1Ra deficiency did not render mice more responsive to systemic exposure of MDP. Despite the increased production of IL-1R agonists IL-1α and IL-1β in response to intraocular injection of MDP, deficiency in IL-1Ra did not predispose mice to MDP-triggered uveitis, albeit intravascular cell rolling and adherence were exacerbated. NOD2 expression was dispensable for the potential of IL-1 to elicit uveitis. However, we find that IL-1Ra does play an important protective role in arthritis induced locally by MDP injection in the joint. Our findings highlight the complexity of NOD2 activation and IL-1 signalling effects that can be compounded by local environmental factors of the target organ. These observations may impact how we understand the molecular mechanisms by which NOD2 influences inflammation of the eye versus joint, and consequently, treatment options for uveitis versus arthritis.

Synergistic effect of muramyl dipeptide with heat shock protein 70 from Mycobacterium tuberculosis on immune activation

Heat shock protein 70 from Mycobacterium tuberculosis (Mtb Hsp70) has been known to modulate immune response including dendritic cell activation. Muramyl dipeptide (MDP) is an immunoreactive derivative of peptidoglycan from all Gram-negative and Gram-positive bacteria and recognized to be responsible for function of Freund's complete adjuvant. In this study, we evaluated effect of MDP on in vitro activation of bone marrow derived dendritic cells (BMDCs) and in vivo production of cytokines and chemokines induced by Mtb Hsp70.MDP treatment with Mtb Hsp70 dramatically increased production of IL-6, IL-12p40 and TNF-α in BMDCs compared with Mtb Hsp70 alone whereas these effects were abolished in Nod2-deficient BMDCs. Phosphorylation of IκB-α and ERK and impairment of phagocytosis, which is an indicator of DC maturation were enhanced by MDP co-treatment with Mtb hsp70 in BMDCs. In addition, ability of Mtb Hsp70-stimulated BMDCs to induce IFN-γ productions of T cells was increased by MDP co-treatment. Finally, intraperitoneal injection of MDP with Mtb Hsp70 dramatically increased production of IL-6, CXCL-1 and CCL2 in serum compared with Mtb hsp70 injection.Our study showed the synergistic effects of MDP with Mtb Hsp70 on DCs and in vivo immune activation. The use of MDP with Mtb Hsp70 to induce immune activation may provide an effective strategy for vaccination to treat cancer and protect against pathogens.

Nucleotide Oligomerization Domain-2 (NOD2)-Induced Uveitis: Dependence on IFN-γ

Nucleotide oligomerization domain-2 (NOD2) plays an important role in innate immunity to sense muramyl dipeptide (MDP), a component of bacterial cell walls. Notably, NOD2 is linked to eye inflammation because mutations in NOD2 cause a granulomatous type of uveitis called Blau syndrome. A mouse model of NOD2-dependent ocular inflammation was employed to test the role of a cytokine strongly implicated in granuloma formation, IFN-gamma, in order to gain insight into downstream functional consequences of NOD2 activation within the eye triggering uveitis. Mice deficient in IFN-gamma, NOD2, or CD11b and their wild-type controls were treated with intravitreal injection of MDP in the presence or absence of IFN-gamma. IFN-gamma production in the eye was measured by ELISA. The intravascular inflammatory response within the iris was quantified by intravital microscopy. NOD2 activation resulted in the production of IFN-gamma within the eye. Deficiency in IFN-gamma diminished the development of MDP-induced uveitis, indicating its crucial role in downstream inflammatory events triggered by NOD2. Moreover, exogenous IFN-gamma markedly exacerbated MDP-induced ocular inflammation in a NOD2-dependent mechanism. The potential of IFN-gamma to enhance inflammation required the adhesion molecule CD11b because CD11b-deficient mice failed to show the synergistic effects of IFN-gamma and MDP cotreatment on adhering and infiltrating cells. IFN-gamma was identified as a downstream mediator of NOD2-driven inflammation and the capacity of IFN-gamma in vivo to enhance the inflammatory potential of NOD2 was demonstrated. Extrapolation of these findings in mice suggests that the dysregulation of IFN-gamma may occur in patients with Blau syndrome, thereby contributing to the granulomatous nature of the disease.

NOD2, the Gene Responsible for Familial Granulomatous Uveitis, in a Mouse Model of Uveitis

NOD2 plays an important role in the recognition of intracellular bacteria through its ability to sense the components of bacterial peptidoglycan (PGN), namely muramyl dipeptide (MDP) and muramyl tripeptide (MTP). Specific mutations in the human NOD2 gene cause Blau syndrome, an autosomal dominant form of uveitis, arthritis, and dermatitis. As a first step toward understanding the role of NOD2 in the pathogenesis of uveitis, the authors developed a mouse model of MDP-dependent uveitis. BALB/c mice and mice deficient in L-selectin or NOD2 received intravitreal injection of MDP, MTP, or PGN. The intravascular response within the iris and cellular infiltration was quantified by intravital microscopy and histologic assessment. MDP induced an acute, ocular inflammatory response, wherein rolling and adhering leukocytes within the vasculature were significantly increased within 6 hours after MDP treatment. A minor increase in cellular infiltration occurred at 12 hours after MDP treatment. The adhesion molecule L-selectin participated in MDP-induced vascular inflammation because L-selectin knockout mice showed a significant decrease in the number of rolling cells. Importantly, NOD2 plays an essential role in ocular inflammation induced by MDP, as indicated by the fact that uveitis did not develop in Nod2 knockout mice in response to MDP. Nod2 knockout mice also showed abolished ocular inflammation in response to MTP but not to PGN treatment. These findings demonstrate a novel mouse model of uveitis, wherein NOD2 plays an essential role in inflammation induced by the minimal components of PGN. Thus, innate immune responses mediated by NOD2 may participate in the development of uveitis in response to bacterial products.

Growth failure after recurrent fever in young guinea pigs

Infection causes fever and suppression of appetite, a combination of effects which threatens normal growth in infected children. We have used an animal model to study the effects on growth of recurrent simulated Gram-positive bacterial infection. After weaning, 10 guinea pig pups underwent surgery under general anaesthesia for the implantation of temperature-sensitive radiotelemeters and thereafter were assigned to receive intramuscular injections of either 50 μg/kg muramyl dipeptide (MDP), or sterile saline. During a 30-day period corresponding to their rapid growth phase, the pups were given eight injections. MDP resulted in fevers of about 1.5 °C on each occasion, but no significant change in body temperature occurred after saline injections. Food intake was suppressed during each febrile episode such that 24-h intake was significantly lower on days of injections of MDP, compared to days between MDP injections in the same animals, and compared to that of animals injected with saline. The rate of weight gain of the MDP-injected guinea pigs was significantly lower than that of the control group and failed even to achieve a rate similar to the saline-injected group in their more adult-like growth phase. Plasma zinc concentration was significantly lower in MDP-compared to saline-injected animals sampled 8 days after the last injection. Our results show that recurrent fever during the growth phase of young guinea pigs results in irreversible growth failure, and that reduced food intake on days when the animals were febrile was at least partly responsible for this reduced rate of growth.

Circulating Inflammatory Mediators during Start of Fever in Differential Diagnosis of Gram-Negative and Gram-Positive Infections in Leukopenic Rats

Gram-negative and gram-positive infections have been considered the most important causes of morbidity and mortality in patients with leukopenia following chemotherapy. However, discrimination between bacterial infections and harmless fever episodes is difficult. Because classical inflammatory signs of infection are often absent and fever is frequently the only sign of infection, the aim of this study was to assess the significance of serum interleukin-6 (IL-6), IL-10, macrophage inflammatory protein-2 (MIP-2), procalcitonin (PCT), and C-reactive protein (CRP) patterns in identifying bacterial infections during start of fever in normal and cyclophosphamide-treated (leukopenic) rats following an injection of lipopolysaccharide (LPS) or muramyl dipeptide (MDP) as a model for gram-negative and gram-positive bacterial infections. We found that, compared to normal rats, immunosuppressed animals exhibited significantly higher fevers and lesser production of all mediators, except IL-6, after toxin challenge. Moreover, compared to rats that received MDP, both groups of animals that received an equivalent dose of LPS showed significantly higher fevers and greater increase in serum cytokine levels. Furthermore, in contrast to those in immunocompetent rats, serum levels of IL-6 and MIP-2 were not significantly changed in leukopenic animals after MDP injection. Other serum markers such as PCT and CRP failed to discriminate between bacterial stimuli in both groups of animals. These results suggest that the use of the analyzed serum markers at an early stage of fever could give useful information for the clinician for excluding gram-negative from gram-positive infections.

Role of the hepatic function in the development of the pyrogenic tolerance to muramyl dipeptide.

We have demonstrated that the hepatic function may have an important role in the development of tolerance to the pyrogenic effect induced by endotoxin. To further investigate if the role of the hepatic function in the development of tolerance also extends to that induced by other pyrogenic stimuli, we investigated the effect of galactosamine, a specific inhibitor of the hepatic protein synthesis, on the development of tolerance to the pyrogenic effect induced by muramyl dipeptide (MDP) in rats. Pyrogenic tolerance was observed after the second intravenous or intraperitoneal injection of MDP (500 microgram/kg), 24 h after the first injection, similar to what was observed with endotoxin. Pyrogenic tolerance was abolished when galactosamine (300 mg/kg ip) was injected simultaneously with MDP (500 microgram/kg iv) on the first day. When uridine (600 mg/kg ip) was administered simultaneously with galactosamine (300 mg/kg ip) and the first injection of MDP (500 microgram/kg ip), pyrogenic tolerance was again observed after the second injection of the peptidoglycan. In conclusion, the hepatic function may not be important only for the development of tolerance to endotoxin, but also to a totally different pyrogenic stimulus such as MDP.

Complement does not mediate the febrile responses of guinea pigs to muramyl dipeptide and polyriboinosinic–polyribocytidylic acid

The complement (C) system and Kupffer cells (Kc) play critical roles in the febrile response of guinea pigs to i.p.-, but not i.v.-injected lipopolysaccharide (LPS). We have investigated whether C and Kc may also mediate this species’ febrile response to muramyl dipeptide (MDP) and polyriboinosinic–polyribocytidylic acid (poly I:C), two other exogenous pyrogens. Both i.v. and i.p. injections of MDP (150 μg/kg) caused initial, transient falls (0.6°C after 90 min by both routes), then rises (0.8°C at 220 and 0.5°C at 200 min, respectively) in core temperature ( T c). Cobra venom factor (CVF)-induced reductions in C variably affected these courses, but the fever indices and the reductions in serum C activity were not correlated. In addition, neither the i.v. nor the i.p. injection of MDP per se induced a significant change in the serum C activity. The i.v. injection of poly I:C (30 μg/kg) caused a 0.6°C T c rise which started within 30 min, peaked at 60 min, and recovered in 5 h. The i.p. injection of poly I:C (50 μg/kg) caused a fever similar to that produced by the i.v. injection, but with a later onset, starting at 90 min after drug administration. Again, C reduction did not affect the overall fever indices, although some individual responses were slightly attenuated. The i.v. injection of poly I:C per se also did not affect the serum C activity. These results indicate that, in contrast to its role in i.p. LPS-induced fever, C is not involved in the febrile responses of guinea pigs to both i.v. and i.p. MDP or poly I:C.

Intracameral Muramyl Dipeptide-Induced Paracellular Permeability Associated with Decreased Glutamate Transporter and g -Glutamyltranspeptidase Activities

Muramyl dipeptide (MDP) (N -acetylmuramyl- l -alanyl- d - isoglutamine) was injected intracamerally to test if MDP applied to the aqueous side of the blood-aqueous barrier would increase paracellular permeability in association with diminished uptake of glutamate. The symptoms of anterior uveitis, i.e., increase in vascular dilatation, could be detected as early as 30 min post MDP injection while aqueous protein concentration did not increase at this time suggesting an initial dissociation between the circulatory and epithelial barrier responses. However, at 45 min, the aqueous protein concentration increased 10-fold (201±174 to 2094±1835 μgml−1;P<0.001) rising progressively to 20-fold above the control eye at 60 min post injection (254±194 vs. 5038±2514 μg ml−1;P<0.001). Epithelial cell barrier paracellular permeability increased at 45 min as evidenced by the enhanced efflux of radiolabelled l -glucose out of the aqueous (8% and 13% faster than control at 45 and 60 min post MDP injection, respectively), coinciding with the accelerated protein influx. A near 50% reduction in efflux of both radiolabelled glutamate and d -aspartate was consistent with reduced glutamate uptake by the transport system X−AG. In addition, a 24% decline in aqueous glutamate, but not aspartate, was detected in the aqueous of the MDP-treated eyes in association with a 54% decrease in iris/ciliary body γ-glutamyltranspeptidase activity consistent with reduced de novo glutamate formation from glutamine. The aqueous of MDP injected eyes also had 6-fold and 34-fold higher prostaglandin E2and F2αconcentrations, respectively (P⩽0.03) as well as reduced AH bicarbonate concentration. These results suggest that increased paracellular permeability is associated with diminished γ-glutamyltranspepidase-mediated glutamate production, X−AGtransport activity, and cellular acidosis in the MDP-induced prostaglandin-mediated inflammation.

Neutralization of pyrogen-induced tumour necrosis factor by its type 1 soluble receptor in guinea-pigs: effects on fever and interleukin-6 release.

1. A soluble form of the tumour necrosis factor (TNF) type 1 receptor (referred to as TNF binding protein, TNF-bp) at a dose of 1 mg per animal, or an equivalent volume of solvent, was injected together with 10 microg kg-1 lipopolysaccharide (LPS) or 50 microg kg-1 muramyl-dipeptide (MDP) directly into the arterial circulation of guinea-pigs and the effects on circulating TNF or interleukin-6 (IL-6) and on abdominal temperature were studied. 2. At 15 or 60 min after injection, LPS-induced and MDP-induced circulating TNF was below the detection limit of the assay and thus completely neutralized in animals treated with TNF-bp. In the control group, TNF was still below the limit of detection in most animals 15 min after LPS was injected; in some animals small traces of TNF could already be detected at that time. However, 60 min after administration of LPS, large amounts of TNF (19508 +/- 4682 pg ml-1) were measured in the control group. MDP-induced TNF in plasma was below the limit of detection 15 min after MDP was injected, and rose to 10862 +/- 3029 pg ml-1 60 min after injection. 3. Low levels of circulating IL-6 (20-40 international units (IU) ml-1) were measured in all groups of animals 15 min after injection of LPS or MDP. This value corresponds to the baseline activity of IL-6 in plasma of guinea-pigs. One hour after administration of LPS, IL-6 rose to 5442 +/- 1662 IU ml-1 in the control group and to a significantly lower value of 1485 +/- 179 IU ml-1 in guinea-pigs treated with TNF-bp. One hour after injection of MDP, circulating IL-6 was 2614 +/- 506 IU ml-1 in the control group, while the corresponding value in animals treated with TNF-bp again was significantly lower (873 +/- 312 IU ml-1). 4. The second phase of the characteristic biphasic LPS fever in guinea-pigs was significantly attenuated in animals treated with TNF-bp. The shorter first phase of the febrile response to LPS was identical in both groups of animals. 5. The late phase of MDP-induced fever (7-22 h after injection) was depressed by treatment with TNF-bp, while the first phase of MDP-induced fever (0-7 h after injection) was significantly enhanced by the neutralization of TNF by TNF-bp.

Fever suppression by subdiaphragmatic vagotomy in guinea pigs depends on the route of pyrogen administration.

It has recently been shown that subdiaphragmatic vagotomy blocks some of the effects of inflammatory stimuli on brain-controlled functions. Therefore, vagal afferent fibers have been proposed to play a prominent role in the communication pathways between the immune system and the brain. In the present study, we investigated the effect of subdiaphragmatic vagotomy on fever induced by intraperitoneal or intramuscular injection of lipopolysaccharide (LPS) or muramyl dipeptide (MDP), which are both cytokine-inducing agents in guinea pigs. Intraperitoneal and intramuscular injections of LPS or MDP were tested in the same animal with an interval of 1 wk. In one experiment, intraperitoneal injections of LPS or MDP were performed at the beginning, followed by intramuscular injections 1 wk later. In another experiment, intramuscular injections of LPS or MDP were performed at the beginning, followed by intraperitoneal injections 1 wk later. The febrile response to intraperitoneal injection of LPS was almost completely abrogated in vagotomized animals compared with sham-operated controls (from 30-330 min after intraperitoneal injection of LPS). The suppression of LPS fever was quantitatively the same in both experiments. In contrast, the response to intramuscular injection of LPS was the same in vagotomized and sham-operated guinea pigs in each of the experiments. Fever induced by intraperitoneal injection of MDP was partly attenuated by subdiaphragmatic vagotomy (between 150 and 300 min after intraperitoneal injection of MDP) in both experiments. Again, the febrile response to intramuscular injections of MDP was not significantly altered by subdiaphragmatic vagotomy. In conclusion, the suppressive effect of subdiaphragmatic vagotomy on fever induced by peripheral immune stimuli depends on the route of pyrogen administration. Because the febrile response to intraperitoneal injection of bacterial pyrogens is strongly diminished in vagotomized guinea pigs, it is suggested that vagal afferent fibers play a crucial role in the transduction of immune signals from the abdominal cavity to the brain.

Effects of ambient temperature on thermoregulation during fever in cats.

Effects of ambient thermal loads on thermoregulatory responses and on the shape of fever curves were studied during muramyl-dipeptide-(MDP)-induced fever in cats. They were implanted with electrodes for chronic recording of electromyograms and cortical and subcutaneous temperatures. A febrile response was induced following intravenous injection of muramyl dipeptide. (1) During the initial phase of fever, the recruitment of heat gain responses and suppression of heat loss responses did not depend on the ambient temperature (Ta). (2) The relative contribution of each thermoregulatory mechanism varied with Ta. (3) Ta did not affect the amplitude of the first febrile peak. However, a second peak in body temperature seen in MDP-induced fever was absent in warm Ta of 30 and 37 degrees C.

Brain regulatory system for the immune response: immunopharmacology and morphology.

On the basis of our previous investigations concerning the influence of some central nervous system structures on the immune reaction, we have shown the existence of a brain regulatory system for immune response (BRSIR). We have investigated this BRSIR using small electrolytic lesions placed in different brain areas of male rats, followed by two methods to evaluate the effect of the lesions on the immune response, the method of delayed skin hypersensitivity and the method of utilization of 3H-thymidine for synthesis of DNA after injection of muramyl dipeptide, a very potent immunostimulating compound. The experiments were performed on rats of the Wistar strain. Lesions were placed from the spinal cord through the brain stem to the cerebral cortex. The results suggest that the following structures constitute parts of the BRSIR: medial frontal cortex (areas Cg 1-3), subnucleus basomedialis and centralis of the amygdala, subnucleus medialis and dorsolateralis of the nucleus parabrachialis, lateral reticular formation (nucleus parvocellularis--mainly areas corresponding with aminergic groups A1-7), part of the raphe reticular formation (nucleus raphealis dorsalis and nucleus linearis--mainly areas corresponding with serotoninergic groups B6-8), and the spinal cord.

Modulation of lipopolysaccharide-induced cytokine gene expression in mouse bone marrow-derived macrophages by muramyl dipeptide.

Previous studies have shown that an i.v. injection of muramyl dipeptide (MDP) before a LPS challenge strongly potentiated serum TNF and IL-6 release in mice. Therefore the direct action of MDP was examined on TNF-producing cells, namely in macrophages stimulated or not by LPS. The level of TNF-alpha, IL-1 alpha, and IL-6 mRNA was determined in bone marrow-derived macrophages (BMM). A marked TNF-alpha mRNA accumulation was found between 1 and 6 h after stimulation with MDP or LPS. LPS-induced IL-1 alpha mRNA transcript was delayed (3 h) than those after MDP induction (1 h). Conversely, kinetic induction of the IL-6 mRNA transcript was delayed in MDP-treated BMM as compared with LPS-stimulated cells. MDP pretreatment of BMM for 3 h not only enhanced the total level of LPS-induced TNF-alpha, IL-1 alpha, and IL-6 mRNA (respectively 2.9-, 1.6-, and 2.4-fold increase), but it also delayed the kinetics of IL-1 alpha and IL-6 species accumulation. The enhancement induced by MDP pretreatment at the level of cytokine mRNA accumulation was correlated with an increase in LPS-induced TNF and IL-6 biologic activity production in supernatant fluids. In addition, in BMM from C3H/Hej mice MDP pretreatment enhanced the weak effect of LPS on TNF mRNA transcript accumulation and was required to produce LPS-induced TNF bioactivity. Our results suggest that MDP and LPS could act through distinct pathway(s) to induce cytokine gene expression. Moreover, the priming effect displayed by MDP could result in modulation of the LPS-induced cytokine gene expression at the transcriptional and/or post-transcriptional level.