Role of interleukin 2 on enhancement of concanavalin A-induced human peripheral blood lymphocyte proliferation by murine B cell mitogens

Murine B cell mitogens such as bacterial lipopolysaccharide (LPS), butanol-extracted water soluble adjuvant (Bu-WSA), dextran sulfate (DS), synthetic muramyl dipeptide (MDP), and its analog MDP-Lys (L18) do not show any mitogenic ability in vitro on human peripheral blood lymphocytes or mixed cell populations of purified T and B cells obtained from the lymphocytes in an ordinary culture system. However, these mitogens are capable of enhancing the mitogenic effect of concanavalin A (Con A) in the cultures. In the presence of one of these mitogens, the activity of interleukin 2 (IL 2), but not interleukin 1, in the supernatants obtained from cultures containing Con A-stimulated T cell and B cell populations was higher than that of control cultures. The role of the newly produced IL 2 in the synergistic effect of the mitogens in human lymphocyte cell cultures was discussed.     

Effect of a synthetic adjuvant on the induction of primary immune responses in T cell-depleted spleen cultures.

N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) stimulates in vitro primary immune responses to SRBC in T cell-depleted (nude) spleen cultures. The stimulation of immune responses by muramyl peptide was antigen dependent. A microculture system was used to compare the T cell-replacing activities of several structural analogues of muramyl dipeptide and to compare the activity of muramyl dipeptide to helper T cells. In a limiting dilution analysis with excess helper T cells or muramyl dipeptide, the frequency of B cell precursors that respond to SRBC was similar, ranging from 1.5 to 5 X 10(-5). Decreasing the cell density in microcultures did not affect the efficiency of B cell precursor responses in the presence of muramyl dipeptide. Muramyl dipeptide was examined for mitogenic activity in spleen cell cultures. In serum-free medium, muramyl dipeptide stimulates slight (3-fold) increases in DNA synthetic activity. In medium supplemented with 5 to 20% fetal calf serum, muramyl dipeptide showed no significant mitogenic activity. There are a number of possible explanations for the T cell-replacing activity of muramyl dipeptide. The most likely is that muramyl dipeptide interacts directly with B cells to mimic the helper T cell signal in the inductive stimulus.     

Determination of mitogenic properties and lymphocyte target sites of Dolichos lablab lectin (DLA): comparative study with concanavalin A and galactose oxidase cell surface receptors

A mannoside-directed lectin has been isolated and purified from the seeds of Dolichos lablab L. by affinity chromatography. We have established that this glycoprotein, which displays high erythroagglutinating activity without blood group specificity, highly activates murine T lymphocytes, and we have described for the first time its mitogenic properties. Although its main properties are close to those of concanavalin A (Con A), the well-known mannoside-directed mitogen devoid of sugar moiety, several differences were found in some of the early events triggered by the two lectins during lymphocyte mitogenic stimulation: higher level of interleukin-2 (IL-2) synthesis, optimal dose for IL-2 synthesis at suboptimal mitogenic concentration, lack of ecto-5' nucleotidase inhibition, and lack of mitogenic inhibition at high lectin concentration. Because the two lectins did not act on the cell surface in exactly the same way, we have compared their receptors involved in mitogenesis on the plasma membrane of murine lymphocytes. We had previously established that the polyclonal activation of these cells probably occurred through high-molecular-weight receptors (200-230 kDa). Since the mitogenic stimulation of lymphocyte by galactose oxidase (GO), like that of Con A, was inhibited by DLA, we analyzed the cell surface receptors that were common to these three polyclonal mitogens. After labeling the neuraminidase/GO-treated cell surface glycoproteins with NaB3H4, we immunoprecipitated the Con A and DLA receptors which are the target of GO mitogenic action. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the precipitates demonstrated that there exist on the lymphocyte cell surface receptors common to the polyclonal mitogens DLA, Con A, and GO. Because Con A and DLA sterically inhibit GO mitogenic stimulation, the common glycoproteins which represent the necessary sites of oxidative mitogenic action are probably those which are involved in DLA and Con A-triggered mitogenesis, despite the different properties of the two lectins. These differences could be explained by the lower molecular weight receptors of the two lectins which are not identical.     

Components of mycobacteria and muramyl dipeptide with adjuvant activity induce lymphocyte activating factor

Several components of mycobacteria including a water-soluble extract (WSA) and an interphase material (IPM) as well as the synthetic cell wall analog muramyl dipeptide (MDP) all stimulated human mononuclear cells (MNL) to produce a factor which was mitogenic for murine thymocytes. The mediator induced by MDP is probably lymphocyte-activating factor (LAF) because it was produced by adherent but not nonadherent MNL and yields two characteristic peaks of activity in the 16,000–22,000 and 60,000–70,000 molecular weight range when eluted from Bio-Gel P-100 columns. The 6-O-stearoyl derivative of MDP was an active inducer of MNL LAF production, whereas, the d-alanine analog of MDP was somewhat less potent. Unfractionated as well as adherent, but not nonadherent, mouse peritoneal cells also produced LAF in response to WSA, IPM, and MDP. P388D₁ cell line macrophages, which are completely devoid of lymphocytes, could be stimulated by WSA, IPM, and MDP to produce LAF after prolonged incubation. These adjuvants did not stimulate nonadherent Balb/C or human blood cells to produce a mitogenic factor. However, when the P388D₁ macrophages were stimulated with these adjuvants in the presence of nonadherent murine or human peripheral blood cells, a mitogenic activity was produced in a shorter period of incubation suggesting that activated lymphocytes can facilitate the production of LAF by macrophages.     

The lack of generalized immunosuppression in C57BL/6 mice during progressive growth of syngenic T lymphoma EL-4 and Lewis lung carcinoma 3LL

The immune competence of C57Bl/6 mice implanted with EL-4 lymphoma of Lewis Lung carcinoma 3LL was investigated during 3 weeks after implantation. Splenic lymphocyte responses to mitogens (Con A, PHA, LPS, PWM) cytotoxic T lymphocytes (CTL) and interleukin-2 (IL-2) production were assessed. A dramatic reduction in mitogenic responses to Con A and PHA was observed during tumour progression. LPS and PWM responses were less depressed. Con A-induced IL-2 production correlated with Con A and PHA responses. Allospecific CTL response to mastocytoma P 815 was not decreased in syngeneic tumour-bearing mice.     

Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP

Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.     

Tumor necrosis factor and its induction by bacterial endotoxins

Using suitable immunomodulators (Corynebacterium parvum vaccine, Zymosan or muramyl dipeptide), lipopolysaccharides (LPS) from various members of the family Enterobacteriaceae (Escherichia, Salmonella, Serratia, Shigella) were tested on rabbits in relation to the production of tumor necrosis factor (TNF). TNF was determined by means of the serum titration of L-929 cell cultures in the presence of Actinomycin D, this with resulting titres of 3.2 x 10(3) to 5.1 x 10(4) IU TNF/ml. Analogous titres were noted after the action of denatured LPS (ie LPS subjected to alkaline hydrolysis or H2O2).     

Tumor cytotoxicity and interleukin 1 production of blood monocytes of lung cancer patients

Summary The effects of lung cancer on the abilities of blood monocytes to produce interleukin-1 and to mediate antitumor activity were examined. The functional integrity of blood monocytes was determined by their capacity to respond in vitro to a variety of activating agents and become tumoricidal, as assessed by a radioactive release assay and ability to produce interleukin-1 in vitro. The results show that the presence of lung cancer significantly increased the number of harvested blood monocytes and that the spontaneous tumoricidal activity of these monocytes was slightly high as compared to monocytes obtained from healthy donors. The production of interleukin-1 by monocytes of healthy donors and lung cancer patients was similar. Blood monocytes obtained from lung cancer patients were less cytotoxic against allogeneic A375 melanoma cells as compared with those of healthy donors subsequent to incubation with a soluble muramyl dipeptide analog or lipopolysaccharide, but were as tumoricidal as those from healthy donors when activated with lipophilic muramyl tripeptide (MTP-PE) entrapped in multilamellar liposomes. The finding that monocytes of patients with lung cancer can respond to MTP-PE encapsulated in liposomes, recommends the use of these liposomes in therapy of human lung cancer.     

β2-Adrenergic agonists bias TLR-2 and NOD2 activated dendritic cells towards inducing an IL-17 immune response

This study tested the hypothesis that activation of β2-adrenoceptors on DCs influences NOD2 signaling along with its cross-talk with Toll-like receptor-2 resulting in altered Th cell priming ability. Th17 cells are a newly discovered lineage of CD4(+) T cells involved in defense against extracellular bacteria and also implicated in autoimmune disorders. Initiation and polarization of the adaptive immune response is controlled by innate immune recognition mediated by DCs. Previous studies demonstrated that adrenergic receptors modulate cytokine production by DCs and affect their Th cell priming ability. We show that the β2-adrenoceptor agonist salbutamol enhanced IL-6 production in murine bone marrow-derived DCs stimulated with the nucleotide-binding oligomerization domain 2 ligand muramyl dipeptide. However, when the Toll-like receptor-2 ligand Pam3CysSK4 was added, salbutamol inhibited IL-12 but did not alter IL-6 and IL-23 expression. Gene expression analysis showed that salbutamol inhibited the p40 subunit as well as IL-12p35, while IL-23p19 and IL-6 were stimulated. Therefore, β2-adrenoceptors modulated cytokine production resulting in a Th17 cell priming cytokine pattern. Indeed, when antigen-pulsed DCs stimulated by muramyl dipeptide or Pam3CysSK4+muramyl dipeptide in the presence of salbutamol were used for in vivo immunization, the resulting Th17/Th1 cell ratio was increased as evaluated by IL-17 and IFN-γ production. In addition, intradermal injection of norepinephrine along with Pam3CysSK4+muramyl dipeptide increased the Th17 response to an immunogenic protein and this effect was reversed by a β2-adrenoceptor antagonist. Thus, β2-adrenoceptors may be involved in the regulation of defense against extracellular bacteria and the pathogenesis of inflammatory diseases.     

Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease

NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or D-isoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.     

Immunobiological properties of a concanavalin A derivative

A monovalent subunit of concanavalin A (Con A) was tested for mitogenic effects on murine splenic lymphocytes in vitro. In contrast to the effects of intact Con A, the monovalent derivative was not mitogenic at any concentration tested. Furthermore, prior exposure of splenic lymphocytes to monovalent Con A rendered the cells refractory to stimulation by the unmodified lectin.     

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

Abstract 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.     

Effect of cisplatin, lipopolysaccharide, muramyl dipeptide and recombinant interferon-gamma on murine macrophages in vitro. II. Production of H2O2, O2- and interleukin-1

Murine macrophage monolayers treated with cisplatin, lipopolysaccharide (LPS), muramyl dipeptide (MDP) or recombinant interferon-gamma (rIFN gamma) for 2-48 h showed significant increases in the release of H2O2, O2- and interleukin-1 (IL-1) as compared to untreated macrophages. The treatment of macrophages with different combinations of the above agents did not induce synergistic or additive effects on the production of H2O2, O2- and IL-1. The priming of macrophages with rIFN gamma had a significant effect in the additional increased production of H2O2, O2- and IL-1 when subsequently treated with cisplatin, LPS or MDP.     

Ontogeny of T-cell mitogen response in Lewis rats. III. Juvenile adherent suppressor cells block adult mitogen responses

Spleen cells from suckling female Lewis rats (4 to 20 days old) were able to suppress mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) of spleen or thymus cells from adult female Lewis rats and thymus cells from suckling Lewis rats. Thymus cells from suckling rats were unable to suppress adult spleen cell mitogenic responses to Con A. Removal of carbonyl iron (cFe)-, plastic-, or nylon-wool-adherent cells removed the suppressive action of juvenile spleen cells, but irradiation did not. Separated plastic-adherent spleen cells from suckling animals suppressed adult mitogenic responses to Con A. at optimal Con A doses 2-mercaptoethanol (2-ME, 2 X 10(-5) M) abolished the suppressive effect of juvenile cells, however, at the hyperoptimal dose of Con A (125 micrograms/ml) even higher doses of 2-ME did not relieve suppression by juvenile cells. These suppressor cells in suckling pups were affected by early weaning which decreased suppression, resulting in enhanced mitogenic responses of juvenile cells and removal of the ability to suppress adult mitogenic response.     

Enhancement in anti-Semliki Forest virus activity of ds RNA by a muramyl dipeptide

Antiviral activity of an interferon-inducing mycoviral ds RNA against Semliki Forest virus infection was considerably enhanced by N-palmitoylmuramyl-L-alanyl-D-isoglutamine (PMDP), a new muramyl dipeptide. This enhancement in activity was not due to increased production of interferon, but resulted probably from a PMDP-induced increase in nonspecific resistance to infection. These results indicate that a combined treatment with an interferon inducer and muramyl dipeptide may prove highly useful to control effectively viral infections.     

Inhibition of macrophage migration by synthetic muramyl dipeptide

N-acetylmuramyl-L-alanyl-D-isoglutamine, a synthetic compound which is known to have a minimal effective structure for an adjuvant activity of cell wall peptidoglycans, was found to inhibit the migration of normal macrophages. It was shown that the inhibition was neither due to cytotoxic or agglutinating effect of the muramyl dipeptide on macrophages nor due to lymphokine production uopn stimulation of lymphocytes by the muramyl dipeptide.     

Highly purified lipoteichoic acid induced pro-inflammatory signalling in primary culture of rat microglia through Toll-like receptor 2: selective potentiation of nitric oxide production by muramyl dipeptide

In contrast to the role of lipopolysaccharide from Gram-negative bacteria, the role of Gram-positive bacterial components in inducing inflammation in the CNS remains controversial. We studied the potency of highly purified lipoteichoic acid and muramyl dipeptide isolated from Staphylococcus aureus to activate primary cultures of rat microglia. Exposure of pure microglial cultures to lipoteichoic acid triggered a significant time- and dose-dependent production of pro-inflammatory cytokines (tumour-necrosis factor-alpha, interleukin-1beta, interleukin-6) and nitric oxide. Muramyl dipeptide strongly and selectively potentiated lipoteichoic acid-induced inducible nitric oxide synthase expression and nitric oxide production. However, it did not have any significant influence on the production of pro-inflammatory cytokines. As bacterial components are recognised by the innate immunity through Toll-like receptors (TLRs) we showed that lipoteichoic acid was recognised in microglia by the TLR2 and lipopolysaccharide by the TLR4, as cells isolated from mice lacking TLR2 or TLR4 did not produce pro-inflammatory cytokines and nitric oxide upon lipoteichoic acid or lipopolysaccharide stimulation, respectively. Lipoteichoic acid-induced glia activation was mediated by p38 and ERK1/2 MAP kinases, as pretreatment with inhibitor of p38 or ERK1/2 decreased lipoteichoic acid-induced cytokine release, iNOS mRNA expression and nitric oxide production. The observed pro-inflammatory response induced by lipoteichoic acid-activated microglia could play a major role in the inflammatory response of CNS induced by Gram-positive bacteria.     

Augmentation of the proliferative response of thymocytes to phytohemagglutinin by the muramyl dipeptide

A synthetic N-acetylmuramyl-l-alanyl-d-isoglutamine or muramyl dipeptide (MDP) and adjuvant-active analogs, but not lipopolysaccharide (LPS), exhibited the augmenting effect on the proliferative response of thymocytes to phytohemagglutinin (PHA). MDP also had a comitogenic effect on PHA-stimulated T lymphocytes. It was shown that the thymocyte-stimulating effect of MDP is not through the production of the monokines by MDP-stimulated macrophages and that MDP has a direct action on lymphocytes.     

Alteration of quaternary structure and biological activity of concanavalin A

A major molecular species of concanavalin A (Con A), a mitogenic lectin from jack bean seeds, has a quaternary structure composed of four homologous subunits and a tetravalent sugar-binding ability. We show that the tetrameric Con A can be converted into a monovalent monomeric form by either photochemical alkylation or hydrogen peroxide/dioxane oxidation of about two tryptophan residues. A divalent dimeric derivative of Con A is also prepared by sulfomethylamidation of about four carboxyl groups. Chemical properties and mitogenic and hemagglutinating activities of these new Con A derivates are compared with those of the tetravalent Con A, as well as of the Con A derivatives that have appeared in the literature on cell biological studies. The significance of the lectin valences in lymphocyte activation and hemagglutination is also discussed.     

Interleukin-6 production in Mycobacterium bovis BCG-infected mice.

Mycobacterium bovis BCG and its subcellular components (bacterial extract, culture filtrate, purified protein derivative, and muramyl dipeptide MDP) are potent in vitro IL-6 inducers in spleen cell cultures from uninfected and BCG-infected BALB/c mice. Both plastic adherent and nonadherent spleen cells are capable of producing IL-6. Athymic nude mice produce more IL-6 than euthymic mice, suggesting that monocyte/macrophages are the main IL-6-producing cells in response to BCG. Finally, IL-6 production seems to be controlled to some extent by T lymphocytes, as down-regulation of CD4+ cells resulted in a marked increase in IL-6 production. Interferon-gamma does not seem to be involved in this regulation.