Selective potentiation of cytokine expression in human whole blood by murabutide, a muramyl dipeptide analogue.

Murabutide is a synthetic muramyl peptide which is in clinical stage of development. Its effect on cytokine production was analysed in human whole blood to reproduce the natural environment. Induced gene transcription within 2 h was associated with the release of cytokines such as tumour necrosis factor (TNF), interleukin-1 beta (IL-1 beta), IL-6, IL-8, and also the anti-inflammatory mediator IL-1ra. This synthesis was not associated with the release of IL-4, IL-12, interferon gamma (IFN-gamma), the three colony-stimulating factors (CSFs) or the soluble TNF receptors. The same series of cytokines were assayed to determine the effect of some recombinant cytokines in association with murabutide. Thus, in the presence of IL-2, IL-6, IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF), the level of cytokines induced by murabutide was enhanced with no change in the other cytokines profile. IL-3 and GM-CSF were more potent in increasing the murabutide-induced response, eliciting synergistic effects on IL-8 and IL-1Ra production, at both the mRNA accumulation and the protein release. Although neither IL-12 nor IFN-gamma were produced in cells stimulated with murabutide alone, some mRNA expression was found with combined treatments. The results indicate that association of murabutide with a cytokine could exert synergistic effects, thus reducing effective doses of the recombinant protein, increasing the release of anti-inflammatory mediators, and triggering efficient cellular immunity.

Selective induction of CD11a,b,c/CD18 and CD54 expression at the cell surface of human leukocytes by muramyl peptides.

Muramyl dipeptide (MDP), murametide, and murabutide which belong to the family of the immunoadjuvant muramyl dipeptides were applied directly to fresh human whole blood and the expression of some surface markers involved in cell adherence in distinct leukocyte populations was investigated. CD11a,b,c/CD18, CD54, CD49d were selected for their involvement in cell adherence, and transferrin receptor (CD71) and low-affinity IgE receptor (CD23) were selected as markers for activated cells. Whereas CD11a was increased only on monocytes, CD11b, CD11c, and CD18 were strongly enhanced on monocytes and polymorphonuclear cells (PMNs) after treatment with MDPs. This increase in membrane expression of integrins, such as CD11b, was not associated with mRNA synthesis, suggesting a mobilization of the CD11b,c/CD18 intracellular pools present in these cells. In contrast, treatment with MDP, murametide, or murabutide enhanced ICAM-1 (CD54) expression on monocyte and PMN cell surface in association with ICAM-1 mRNA synthesis. No variation of CD49d expression was detected on leukocyte surface after incubation with MDPs. Transferrin receptor (CD71) expression and low-affinity receptor for IgE (CD23) expression were increased on monocyte only after incubation with LPS used as positive control. Moreover, no observable change in the selected markers was detected on lymphocyte after MDPs or LPS treatment. These results indicate that MDPs seem to act preferentially on monocytes and PMNs in increasing the level of molecules involved in cellular adhesion process, either in provoking the expression of preformed molecules or in inducing their synthesis. This contributes to understanding the mechanism of the activities of muramyl peptides on specific and nonspecific immunity.

Modulation of expression of class II MHC and CD40 molecules in murine B cells by various muramyl dipeptides.

Several compounds of the MDP (muramyl dipeptide) series which have the capacity to enhance the immune response to antigens exerted a comitogenic effect on murine splenic B cells. The expression of surface class II major histocompatibility and CD40 antigens was used to more accurately evaluate the comparative influence of the synthetic agents on mature B cells and on the pre-B cell line 70Z/3. MDP and two adjuvant-active analogs enhanced expression of both surface molecules and increased the response to lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) in splenic B cells. The three synthetic adjuvants alone did not lead to expression of cell-surface I-Ad or CD40 in 70Z/3 cells, indicating that they were unable by themselves to achieve differentiation of pre-B cells to a mature B cell phenotype. However, they increased the CD40 level induced by treatment with LPS. In this cell line, the response (CD40 protein and mRNA) to IFN-gamma was strongly increased by MDP but not by the two other compounds. Actually, MDP was the only adjuvant among the three compounds to functionally activate the transcription factor NF-kappaB, to induce kappa transcription, and to stimulate surface kappa light-chain expression in 70Z/3 cells. The response to muramyl dipeptides in mature splenic B cells could appear independent of the transcription factor.

Differentiation of murine pre-B cell line by an adjuvant muramyl peptide via NF-kappa B activation.

Muramyl dipeptide (MDP) induces NF-kappa B activation in the murine pre-B cell line 70Z/3, increases the expression of surface immunoglobulins, and potentiates the response to other inducers such as LPS or IL-1. In the present study we investigated whether NF-kappa B activation was related to the MDP-stimulated immunoglobulin expression. In a gel shift assay our results confirmed that MDP but not MDP(D,D), an adjuvant-inactive stereoisomer, could induce a kappa B-binding activity in 70Z/3 cells. The LPS or IL-1 induced NF-kappa B binding activity was increased in the presence of MDP but not of MDP(D,D). A mutant of the cell line called 1.3E2, defective in NF-kappa B activations by LPS, did not respond to MDP. The enhanced surface immunoglobulin expression induced in the wild type 70Z/3 cells by MDP alone or combined to LPS, IL-1 or IFN gamma was not obtained in this variant. The ability of various treatments to activate the kappa gene enhancer was quantitatively evaluated in cells transfected with a kappa-enhancer-luciferase expression plasmid. Treatment of transfected 70Z/3 cells with MDP resulted in a dose-dependent enhancement of luciferase activity, an additive effect to that induced by LPS or IL-1. Treatment of the defective variant transfected with the same construct did not result in luciferase expression after stimulation with the various agents. The transient transfection assays were used to compare the effectiveness of some MDP analogs. Two adjuvant-active compounds unable to enhance kappa light chain expression did not increase the basal response in the transfected 70Z/3 cells, indicating that NF-kappa B activation was not related to the adjuvant potency of MDP but correlated with the kappa induction.

Regulation of lipopolysaccharide-induced tumor necrosis factor production by cyclosporin A in mice primed with muramyl dipeptide.

The effect of cyclosporin A (CsA) on tumor necrosis factor (TNF) or interleukin-6 (IL-6) production was evaluated in vivo in primed or unprimed mice challenged with lipopolysaccharide (LPS). Both pretreatment with BCG infection or with muramyl dipeptide (MDP) prior to LPS challenge resulted in an increase in the cytokine bioactivity level in the blood. CsA administration inhibited the TNF production. In unprimed mice, either normal or sensitized to LPS lethality by galactosamine treatment, a marked decrease in the cytokine level was observed after injection of CsA. After adrenalectomy, the yield of both TNF and IL-6 following LPS injection was markedly elevated but decreased by CsA administration. Ex vivo experiments have shown that the inhibitory effect of CsA could be demonstrated at the level of macrophages from mice previously given the drug. If mice had received MDP, in vitro responses of cells to LPS were enhanced but again CsA decreased the mRNA expression and protein secretion.

Differential role of interferon-gamma in the potentiating effect of muramyl peptides for enhanced responses to lipopolysaccharide in mice: effect of cyclosporin A.

Cyclosporin A (CsA) administration reduced mortality in mice sensitized to endotoxic toxicity by various agents, such as muramyl dipeptide (MDP) or a lipophilic derivative. CsA is an inhibitor of a variety of T cell responses, suggesting that muramyl peptides could influence LPS-induced effects via the release of lymphokine. The potentiation of TNF production by pretreatment with muramyl peptides was comparable in nude mice and in controls, indicating that it is a T-independent mechanism, and CsA produced a similar inhibition in both groups. Neutralizing antibody to IFN-gamma did not change the elevated TNF level obtained in the blood when LPS was given after a muramyl peptide. However, the same treatment with anti-IFN-gamma MAb prevented the death of mice challenged with LPS plus MDP or plus a lipophilic derivative displaying similar effects. In comparing three selected muramyl peptides, we also show that the priming effect could be dissociated from the toxic synergism with LPS.

Differential regulation of surface immunoglobulin expression by various muramyl dipeptides in a murine pre-B cell line.

The murine pre-B cell line 70Z/3 responds to lipopolysaccharide (LPS), interleukin-1 (IL-1) or interferon-gamma (IGN gamma) by kappa gene transcription and expression of surface IgM (sIg). We found that muramyl dipeptide (MDP), a synthetic immunoadjuvant analog of a bacterial membrane structure, produced a weak increase in the number of sIg-positive 70Z/3 cells as measured by immunofluorescence staining. This number was significantly increased after exposure to MDP. Moreover, when MDP was used in combination with LPS, IL-1 or IFN gamma, an enhancement of sIg expression was observed showing an early influence of MDP in the presence of a second stimulant. Unexpectedly, two adjuvant-active analogs of MDP did not share its capacity to stimulate differentiation of the cell line when used alone or associated with other agents, indicating that adjuvanticity of MDP was not the only requirement. Two other products of bacterial origin, a Staphylococcus aureus cell extract (SAC) and the Toxic Shock Syndrome Toxin TSST-1 could neither enhance the kappa gene expression in 70Z/3 cells nor increase the MDP effect. The stimulating effect displayed by MDP could by related to NF-kappa B activation.

Selective modulation of lipopolysaccharide-induced death and cytokine production by various muramyl peptides.

Pretreatment of animals with the adjuvant muramyl dipeptide enhances both the production of circulating tumor necrosis factor and the sensitivity to the lethal effect of a lipopolysaccharide (LPS) challenge. The present study examined the capacity of various adjuvant muramyl dipeptide derivatives to potentiate responsiveness to LPS administration. Cytokine levels in serum were determined at various time intervals after LPS administration by bioassays and immunoassays; the cytokines examined were tumor necrosis factor, interleukin-1, interleukin-6, and gamma interferon. The time course of cytokine response was not modified by the pretreatment, but most of the levels were strongly enhanced. However, of the four compounds which were found to be potent priming agents, only two caused an increased sensitivity to LPS lethality, showing that elevated titers of cytokines in serum were not correlated with host sensitization. Interestingly, previous studies have shown that these two compounds also display neurobiological properties, implying a possible role of the central nervous system in LPS lethality. However, two hydrophilic derivatives with low activity as priming agents were capable of decreasing the toxicity of LPS when given after the challenge in galactosamine-sensitized mice. These results illustrate the diversity of responses elicited by immunological priming. They raise unanswered questions on the importance of endogenous mediators in the pathophysiological alterations during toxic shock.

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.

Differential priming for endotoxin-induced circulating cytokine production by tumor necrosis factor-alpha and interleukin 1 beta.

In unprimed mice, a single injection of a non-lethal dose of lipopolysaccharide (LPS) produced a rise in tumor necrosis factor (TNF) and interleukin 6 (IL 6) activities. Peak serum concentrations were attained, respectively, 1.5 hr and 2.5 hr after the challenge. Pretreatment with recombinant human TNF-alpha (rHuTNF) had a priming effect for enhanced production of both serum cytokines without any change in kinetics. The enhancement was more pronounced in the TNF (15-fold) than in the IL 6 (4-fold) response. Recombinant murine TNF caused a comparable increase in LPS-induced cytokine release. In contrast, comparable pretreatment with another macrophage-derived cytokine, recombinant human interleukin 1 beta (HuIL1-beta), revealed a negative effect on LPS-induced TNF release whereas IL 6 in the blood reached levels similar to those found after priming with rTNF. Moreover, when administered in combination with rHuTNF, rHuIL1-beta inhibited the priming effect on TNF autocrine production.

Biological activity of a new synthetic muramyl peptide adjuvant devoid of pyrogenicity.

Immunostimulant activities of muramyl dipeptide (enhancement of specific immune responses and of nonspecific resistance to infection) were retained by its N-acetylmuramyl-L-alanyl-D-glutaminyl-n-butyl ester derivative, although very large amounts administered intravenously, or even by the very sensitive intracerebroventricular route, did not elicit fever in the rabbit. This analog also appeared to be devoid of other secondary effects which have been observed after administration of muramyl dipeptide.

Enhancement of resistance to infections by endotoxin-induced serum factor from Mycobacterium bovis BCG-infected mice.

Necrosis of a variety of transplanted murine tumors can be induced by serum from Mycobacterium bovis BCG-treated mice challenged with a lethal dose of endotoxin. Results reported here show that the tumor necrosis serum (TNS) enhances resistance to infections, protecting mice against two types of challenges, either with Klebsiella pneumoniae or with the intracellular parasite Listeria monocytogenes. Moreover, TNS activity was demonstrated in animals which are refractory to lipopolysaccharide and very susceptible to infections, such as 8-day-old mice and adult C3H/He mice. Protection passively transferred by TNS was not related to antibodies, since it was not decreased by absorption with homologous organisms.

Immunostimulant activities of a lipophilic muramyl dipeptide derivative and of desmuramyl peptidolipid analogs.

The immunostimulant properties of a new muramyl dipeptide (MDP) derivative bearing a lipophilic moiety on the C-terminal end of the peptide chain are described. It is shown, in particular, that 1,O-(acetylmuramyl-L-alanyl-D-isoglutamine-L-alanyl)-glycerol-3-mycolate had increased immunostimulant activity in comparison with MDP. It induced hypersensitivity even when administered with an antigen in saline, and it gave higher protection against bacterial infections than did MDP. A quite unexpected finding was obtained with the corresponding desmuramyl compound 1,O-(L-alanyl-D-isoglutamine-L-alanyl)-glycerol-3-mycolate, which had no activity in producing humoral antibodies but was just as active as the muramic acid-containing compound in stimulating nonspecific resistance to bacterial infections. It was not pyrogenic. Modifications of the peptide moiety or the lipid moiety of this peptidolipid led to decrease, or even loss, of activity. These results show the importance of the N-acetylmuramyl moiety in MDP for humoral antibody production. The peptidolipid 1,O-(L-alanyl-D-isoglutamine-L-alanyl)-glycerol-3-mycolate is the first member of a new category of nonspecific immunostimulants.