Suppression of allergic reaction by lambda-carrageenan: toll-like receptor 4/MyD88-dependent and -independent modulation of immunity.

BACKGROUND: Recognition of foreign substances by innate immunity through pattern recognition receptors (PRRs) regulates acquired immunity such as allergic reaction. Because PRRs recognize heterogeneous ligands, daily food intake can potentially regulate immune allergic reaction. OBJECTIVE: Elucidation of the effect of lambda-carrageenan on allergic reactions was aimed. METHOD: IFN-gamma and IL-4 was measured in in vitro T cell-stimulated culture. Cytokine production from macrophages in response to lambda-carrageenan was measured as indicator for innate immunity activation. Mice were immunized with OVA in alum to induce specific IgE, and then histamine release was induced by systemic injection of OVA. RESULTS: Activation of innate immunity by lambda-carrageenan is dependent on Toll-like receptor-4 (TLR4) and MyD88, in which induction of pro-inflammatory cytokines such as TNF-alpha and IL-6 was largely impaired in macrophages from TLR4- and MyD88-deficient mice. Footpad oedema, a model for in vivo inflammatory reactions, was significantly reduced in these mice. Similar to recent evidence showing a preference for the stimulation of Th1 via TLR/MyD88 signalling, lambda-carrageenan showed enhanced IFN-gamma and decreased IL-4 in stimulated T cell cultures. Interestingly, increased IFN-gamma production was still seen in TLR4- and MyD88-deficient splenocytes. Oral administration of lambda-carrageenan to immunized mice successfully decreased OVA-specific IgE, and lambda-carrageenan was also effective in previously immunized mice. Further, serum histamine release upon systemic challenge of OVA was significantly inhibited. Neither OVA-specific IgG1/IgG2a nor cytokine secretion from in vitro cultures were altered, suggesting the involvement of multiple PRRs as demonstrated by TLR4/MyD88-independent IFN-gamma up-regulation. The simultaneous feeding of OVA with lipopolysaccharide abrogated oral tolerance, but lambda-carrageenan was not only devoid of such an effect but was also found to promote oral tolerance in the absence of TLR4. CONCLUSION: lambda-Carrageenan was suggested to be a useful dietary supplement to ameliorate allergic reactions while maintaining oral tolerance-dependent intestinal homeostasis.

B-1 B cell IgM antibody initiates T cell elicitation of contact sensitivity.

Although B-1 B cells have received considerable attention, their actual role in the normal functioning of the immune system is unclear. The hypothesized role of B-1 cell IgM in natural protective immunity is just being established. We have uncovered a separate and novel role for B-1 cell IgM in initiating the elicitation of acquired T cell-dependent contact sensitivity (CS), the prototype of in vivo T cell immunity, early after immunization (within 4 days). The recent recognition of a similarly unanticipated role of B cells in a variety of T cell responses, may indicate that B-1 cell IgM has a broader role in immunity than thought previously. We showed that 24 hr CS responses, and rises in local IFN-gamma levels at 24 hrs later after antigen (Ag) challenge the ears, were absent in pan B cell and antibody deficient mice. The mechanism of B cell involvement in CS-initiation is via local C5a generation early (1-2 hrs) after antigen (Ag) challenge of the ears, in 4 day contact sensitized mice. C5a activates local mast cells to release serotonin (5-HT) and TNF alpha to induce endothelial ICAM-1 and VCAM-1, leading to T cell recruitment. We hypothesized that C5a was generated via complement activation due to antibodies forming local AgAb complexes, and that B-1 cell IgM was involved because isotype switching of B-2 cells to produce C-activating IgG isotypes, could not occur as early as day 4. Indeed, B-1 cell deficient CBA/N-xid mice lacked C5a in 2 hr ear extracts, and had impaired CS ear swelling and elaboration of IFN-gamma at 24 hrs. Importantly, adoptive transfer of purified normal peritoneal B-1 cells, or just i.v. injection of Ag-specific IgM monoclonal antibodies in sensitized xid, restored deficient early C5a and late 24 hr ear swelling. These results suggest that early after Ag challenge, specific B-1 cell IgM, produced at distant sites by prior sensitization, forms AgAb complexes that trigger elaboration of C5a, to activate mast cell release of vasoactive TNF alpha and 5-HT to initiate CS, leading to T cell recruitment. We postulate that antibody of various isotypes possibly may lead to local vascular activation to aid in T cell recruitment in a variety of T cell responses, but that very early after immunization, Ag-specific IgM produced by B-1 cells, preferentially serves this important function.

Early local generation of C5a initiates the elicitation of contact sensitivity by leading to early T cell recruitment.

We have shown previously that an early complement C5-dependent cascade is required to recruit T cells to elicit 24-h contact sensitivity (CS) responses. In this paper, we have characterized molecular events of this early required cascade by biochemically analyzing extracts of mouse ears undergoing elicitation of CS. Chemotactic activity was found after local Ag challenge, in CS ear extracts early (by 1 h), in CS ear extracts late (through 24 h), in previously immunized mice, but not in ears of vehicle-immunized or non-immune-challenged mice. The early chemotactic activity at 2 h was likely caused by C5a, because it was neutralized in vitro by anti-C5a Ab, was inactive on C5aR-deficient (C5aR-/-) macrophages, and was absent in C5-deficient mice. The activity was present in T cell-deficient mice, but elaboration was Ag-specific. This T cell-independent, Ag-specific elaboration of C5a early in CS ear responses likely led to T cell recruitment, because subsequent local IFN-gamma mRNA and protein expression, as markers of T cell arrival and activation, began by 4 h after Ag challenge. In contrast to early C5a chemotactic activity, late chemotactic activity 24 h after Ag challenge was unaffected by anti-C5, was active on C5aR-/- macrophages, was T cell-dependent, and by ELISA appeared largely due to chemokines (macrophage-inflammatory protein-1alpha and -1beta, IFN-gamma-inducible protein-10, and monocyte chemoattractant protein-1). Importantly, early generation of C5a was required for T cell recruitment because C5aR-/- mice had absent 24-h CS. Taken together, these findings indicate an important linkage of C5a as a component of early activated innate immunity that is required for later elicitation of acquired T cell immunity, probably by facilitating the initial recruitment of T cells into the Ag-challenged local site in CS responses.

IL-12 is produced by antigen-presenting cells stimulated with soluble alphabeta TCR and restores impaired T(h)1 responses.

Contact sensitivity (CS) is a cutaneous T(h)1 response that is induced by skin painting with reactive hapten. In prior in vivo studies of CS, we showed that recombinant soluble alphabetaTCR (sTCR) acted non-specifically to protect CS-effector T cells from suppression, but no molecular mechanism was determined. In the current study, we employed an in vitro system to investigate the mechanism of how sTCR protect CS-effector T cells from suppression. Immune CS-effector cells and appropriate hapten-conjugated antigen-presenting cells (APC) were incubated together with down-regulatory culture supernatant produced by suppressive spleen cells from mice tolerized i.v. with specific hapten, which produced strong inhibition of IFN-gamma production by the CS-effector cells. Importantly, addition of two different sTCR, of unrelated specificity, reversed this down-regulation and thus restored IFN-gamma production. We found that the APC, and not the CS-effector T cells, were the locus of the sTCR-mediated protection and showed direct binding of sTCR to APC by flow cytometry. Further, addition of anti-IL-12 showed that sTCR protection was due to IL-12 induced by sTCR and released by the APC, and was confirmed by ELISA measurement of IL-12 induced in APC supernatants by sTCR incubation. These results indicated a possible new regulatory loop in which suppression was reversed by IL-12 derived from APC, following direct surface binding of sTCR, and enhanced by IFN-gamma production from the T(h)1 CS-effector cells.

IL-12 reverses established antigen-specific tolerance of contact sensitivity by affecting costimulatory molecules B7-1 (CD80) and B7-2 (CD86).

Cutaneous painting with reactive haptens induces contact sensitivity (CS) responses that are in vivo examples of T cell immunity. In contrast, high dose i.v. administration of the hapten can induce tolerance. We investigated the effect of IL-12 on reversal of this tolerance and attempted to determine in vitro the mechanism of this reversing effect by measuring proliferation and IFN-gamma production by CS effector T cells stimulated with hapten-conjugated APC, and we also measured CS ear swelling in vivo. The in vitro responses of T cells to hapten-APC became absent in tolerized mice, paralleling impaired in vivo CS responses. Addition of IL-12 to cultures manifesting this fully established in vitro tolerance completely restored impaired responses of tolerized T cells. The reversing effects of IL-12 were not blocked by anti-IFN-gamma mAb, but were blocked by mAbs against B7-1, more strongly by anti-B7-2, and by both Abs together. Additional in vivo ear-swelling response experiments confirmed the reversing effects of IL-12 on established tolerance. To examine whether the IL-12 effect depended on stimulation of IFN-gamma, we directly injected IFN-gamma into tolerized mice. This partially mimicked but did not fully reconstitute the effects of IL-12. In summary, IL-12 abrogation of established tolerance of CS may have been partially due to endogenous production of IFN-gamma, but appeared mainly due to direct activation of the tolerized T cells by affecting signaling through costimulatory molecules B7-1 and B7-2.

Required early complement activation in contact sensitivity with generation of local C5-dependent chemotactic activity, and late T cell interferon gamma: a possible initiating role of B cells.

Complement (C) is an important component of innate immunity, and was also shown recently to participate in induction of acquired B cell humoral immunity. In this study, we present evidence that C also participates in acquired T cell immunity. We found that C was involved in early events of the efferent elicitation phase of contact sensitivity (CS), and delayed-type hypersensitivity (DTH). Thus, CS and DTH were inhibited by administration of a C-blocker, soluble recombinant C receptor-1 (sCR1), when given 30 min before, but not 3 h after local antigen challenge. Among C components, local C5 were thought crucial to elicitation of CS, since local administration of anti-C5 monoclonal antibodies or locally injected C-depleting cobra venom factor also inhibited CS and DTH. These findings were consistent with our previous finding of the importance of C5 for CS elicitation, using congenitally C5-deficient mice. To dissect the mechanism of C dependence in CS, we demonstrated that locally increased early macrophage chemotactic activity (probably C5a) in evolving CS skin extracts, as well as late elaboration of IFN-gamma, were both inhibited by anti-C treatment. In addition, histological analysis showed that leukocyte recruitment into CS ear sites was similarly C-dependent. Furthermore, an initiating role of B cell-derived C-fixing immunoglobulin was suggested by demonstration of impaired CS responses in B cell-deficient mice. In summary, these results suggest that C was activated locally, perhaps via a B cell product, in an important early component of the stepwise events necessary to elicit CS, leading to local production of C5-dependent macrophage chemotactic activity and later IFN-gamma, and subsequently leading to cell infiltration, for development of T cell-dependent CS.

Possible involvement of C5/C5a in the efferent and elicitation phases of contact sensitivity.

The elicitation of 24-h contact sensitivity (CS) in mice requires a serotonin-dependent, 2-h response called CS initiation. We studied the role of complement (C) by comparing CS in DBA/1 (C5-normal) vs DBA/2 (C5-deficient) mice and found impaired 2-h, but not 24-h, CS. We showed previously that 2-h responses represent CS initiation and are required for elicitation of 24-h responses. Treatment of C5-deficient mice with absent macroscopic responses (ear swelling) with a selective serotonin antagonist inhibited 24-h CS, suggesting that C5-deficient mice had submacroscopic (i.e., microscopic), serotonin-dependent CS initiation. When normal mouse serum was used as a source of C5 to reconstitute C5-deficient mice, significant 2-h responses were restored. Furthermore, heat treatment of normal mouse serum to inactivate C abrogated restoration of 2-h responses. Thus, C5 was suggested to be involved in CS initiation. Using a suboptimal immunizing dose of Ag revealed an impaired 24-h component of CS in DBA/2 mice, but not in DBA/1 mice, and also in C5-deficient B10.D2/o mice compared with C5-normal B10.D2/n mice with a suboptimal eliciting dose of Ag. Again, reconstitution of B10.D2/o mice with normal mouse serum restored deficient 24-h CS responses. Thus, 2-h and classical 24-h CS probably depend in part on C5. These results imply that C5 may play a role in the elicitation of 24-h CS, probably via required preceding CS initiation.

Anti-inflammatory effects and specificity of L-156,602: comparison of effects on concanavalin A and zymosan-induced footpad edema, and contact sensitivity response.

We investigated the in vivo selective anti-inflammatory effect of L-156,602, which was first identified as a preferential delayed-type hypersensitivity-suppressant in our screening program and first reported to be a C5a antagonist. The agent most profoundly suppressed footpad edema 4 h after elicitation by concanavalin A (con A) and also caused a significantly impaired response after a further 20 h. Footpad edema induced by either serotonin, carrageenan or zymosan was not much influenced by the agent. Although the dominant cell population that migrated in response to con A and zymosan 4 h after elicitation was neutrophils, L-156,602 specifically prevented the con-A-induced migration of neutrophils, suggesting a distinct mechanism of neutrophil recruitment between con A and zymosan-induced inflammation. The agent also reduced the contact-sensitivity response, especially in host mice sensitized with a moderate dose of picryl chloride and almost completely suppressed the infiltration of mononuclear leukocytes and neutrophils into the site of inflammation. These selective effects of L-156,602 on inflammatory reactions appeared to be not merely via C5a antagonism.

Effects of microbial products on glucose consumption and morphology of macrophages.

We studied the effects of microbial products on glucose consumption and morphology of macrophages which were elicited with thioglycollate medium. Macromolecules such as lipopolysaccharide (LPS), tumor promoters, and respiratory inhibitors increased macrophage glucose consumption without inducing evident morphological changes. The assay system was used to screen for active substances in culture broth extracts from actinomycetes. Among them, aureothin increased glucose consumption of macrophages and inhibited respiration of a rat mitochondrial fraction. Concanamycin A induced morphological changes of macrophages into needle-like shapes but not of cloned cells including the macrophage-like cells J774.1. This compound changed fibrosarcoma L929 cells into round shapes without affecting the shape of a nontransformed fibroblast, BALB/3T3 cells. Antimycin and concanamycin A increased tumor-killing activity of macrophages when added during the effector phase. These results suggest that this assay system is simple and sufficiently reproducible and thus usable for screening for modulators of macrophage function among natural products.

Melastin, a novel product of Streptomyces that selectively inhibits leukemia cell growth.

In the course of our screening for new immunomodulators, a novel compound, melastin, was purified from the culture broth of Streptomyces. Melastin was purified through absorption to Diaion HP-20, ethanol precipitation, and anion exchange column as a brown powder. The molecular weight was estimated as 5000 +/- 3000 by gel filtration HPLC. Melastin suppressed lipopolysaccharide (LPS)-induced blastogenesis of B cells more profoundly than concanavalin A (con A)- or phytohemagglutinin (PHA)-induced blastogenesis of T cells. Moreover, it selectively inhibited the growth of several leukemia cells as compared with interleukin-dependent nontransformed leukocytes. No selectivity was observed between nontransformed fibroblasts and their oncogene-transformed variants. Melastin did not selectively inhibit macromolecule synthesis of leukemia cells.

Effects of L-156,602, a C5a receptor antagonist, on mouse experimental models of inflammation.

L-156,602, a C5a receptor antagonist, was found as an immunosuppressant with preferential effects on delayed-type hypersensitivity (DTH) in our screening program and it was shown that L-156,602 suppressed the efferent phase of DTH. Here, we tested its effects on experimental models of inflammation induced in mice. L-156,602 did not suppress serotonin- and carrageenan- induced inflammation while it completely suppressed concanavalin A-induced inflammation 4 h after elicitation. The inflammation appeared 24 h after the elicitation with concanavalin A and it was significantly suppressed by L-156,602. Muramyl dipeptide (MDP)-induced acute joint inflammation was also significantly suppressed by L-156,602. These results demonstrated the unique immunomodulating properties of L-156,602 in mouse experimental models of inflammation.

Preferential suppression of delayed-type hypersensitivity by L-156,602, a C5a receptor antagonist.

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.

Immunomodulating properties of prodigiosin 25-C, an antibiotic which preferentially suppresses induction of cytotoxic T cells.

An antibiotic, prodigiosin 25-C, preferentially suppresses cytotoxic T lymphocytes (CTL) without affecting antibody production. Here, we investigated the effect of prodigiosin 25-C on delayed-type hypersensitivity (DTH), graft versus host reaction (GvHR) and allogeneic skin graft rejection. DTH reactions were markedly inhibited by ip treatment of the mice with prodigiosin 25-C. Cell transfer experiments indicated that prodigiosin 25-C exerted its suppressive effect on the late efferent phase rather than on the induction phase of DTH. Prodigiosin 25-C suppressed induction of anti-host CTL when GvHR was induced by iv inoculating splenocytes of parental C57BL/6 mice to adult unirradiated BDF1 mice. It had little effect on GvHR-induced splenomegaly observed 2 weeks after the inoculation, but significantly delayed the subsidence of splenomegaly as revealed 8 weeks later, suggesting that suppression of CTL converts immunosuppressive GvHR to immunostimulative one as reported by G. M. Shearer. However, reduction of interleukin-2 (IL-2) production and mitogen responses induced by GvHR were not rescued by prodigiosin 25-C treatment. Prodigiosin 25-C moderately prolonged survival of major histocompatibility (MHC)-mismatched skin grafts. Since the mode of action of prodigiosin 25-C is distinct from those of cyclosporin A and FK506, these results demonstrate potential usefulness of the antibiotic for a supplementary immunosuppressant.

Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease.

The neutral protease II (NpII) from Aspergillus oryzae is a zinc-containing metalloprotease with some unique properties. To elucidate its structure, we isolated a full-length cDNA clone for NpII. Sequence analysis reveals that NpII has a prepro region consisting of 175 amino acids preceding the mature region, which consists of 177 amino acids. As compared with other microbial metalloproteases, NpII is found to be unique in that it shares only a limited homology with them around two zinc ligand His residues and that the positions of the other zinc ligand (Glu) and the active site (His) cannot be established by homology. When a plasmid designed to express the prepro NpII cDNA was introduced into Saccharomyces cerevisiae and the transformant was cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), it secreted a proNpII. However, in a culture of the same medium containing 0.2 mM ZnCl2, it secreted a mature NpII with a specific activity and N-terminus identical to those of native NpII. This observation suggests that either an autoproteolytic activity or a yeast protease effected the processing.

Enhanced enzymatic activity and stability of trypsin by reductive alkylation in solid phase.

Amino groups of trypsin (EC 3.4.21.4) were reductively alkylated in solid phase to obtain a surface-active and biologically active enzyme in an o/w emulsion system. Trypsin adsorbed on a benzamidine-sepharose column was reductively alkylated with n-octanal in the presence of sodium borohydride, i.e., trypsin-C8. Activity of trypsin-C8 against Nalpha-benzoyl-L-arginine-p-nitroanilide was three times higher than that of native trypsin. Activities of trypsin and trypsin-C8 against casein were almost the same. After incubating the trypsin solution at 40 degrees C for 1 h, residual activities in the emulsion and solution systems were 64.2 and 57.4%, respectively. On the other hand, residual activities of native trypsin following incubation were 21.8% in the emulsion system and 33.2% in the solution system. Enhancement of trypsin-C8 stability in the emulsion system may derive from interaction between the hydrophobic areas of trypsin-C8 molecules and the hydrophobic phase of the emulsion.

Selective immunosuppression of prodigiosin 25-C and FK506 in the murine immune system.

The immunosuppressive effects of prodigiosin 25-C were studied in comparison with FK506. Both prodigiosin 25-C and FK506 suppressed T cell proliferation in response to concanavalin A (con A) or phytohemagglutinin (PHA) more significantly than that to lipopolysaccharide. However, prodigiosin 25-C inhibited con A-mediated mitogenic response more strongly than PHA-mediated one. FK506 showed no selectivity among those responses. In addition, when higher concentration of con A was used an inhibitory effect of prodigiosin 25-C became more evident whereas that of FK506 became less evident. Furthermore, prodigiosin 25-C affected neither interleukin-2 (IL-2) production nor IL-2 receptor (IL-2R) and transferrin receptor (TF-R) expression in vitro, though FK506 extensively inhibited IL-2 production and significantly suppressed IL-2R and TF-R expression. When comparing the effects of prodigiosin 25-C and FK506 in vivo by injecting antigens of different nature to a mouse, prodigiosin 25-C selectively inhibited cytotoxic T lymphocyte (CTL) activity induced by an allogenic mastocytoma, P815, without affecting production of antibody against a thymus dependent (TD) antigen, sheep red blood cell (SRBC). On the contrary, FK506 significantly inhibited both CTL induction and the antibody production. When Brucella abortus, a thymus independent (TI) antigen, and SRBC were simultaneously challenged to a mouse, neither prodigiosin 25-C nor FK506 affected antibody production against the TI antigen while the effect on the TD antigen were the same as described above. The present results revealed the unique immunosuppressive property of prodigiosin 25-C which was different from that of FK506.

Suppression of cytotoxic T cell induction in vivo by prodigiosin 25-C.

Prodigiosin 25-C (PrG25-C) was discovered as an immunosuppressant in the course of our screening for immunomodulating substances. In this system, PrG25-C inhibited T lymphocytes proliferation and was less suppressive against B lymphocytes. PrG25-C was also a powerful inhibitor of cytotoxic T cell induction by mixed lymphocyte reaction and completely suppressed induction of H-2 specific cytotoxic cells at 12.7 nM. PrG25-C also inhibited in vivo induction of H-2 restricted cytotoxic T lymphocytes at a dose of 0.5 mg/kg but had little myelotoxicity because numbers of blood leukocytes and splenocytes of PrG25-C-treated mice were comparable to those of nonsensitized mice. No inhibitory effects of PrG25-C were observed on the production of anti-SRBC antibody. These results indicate that PrG25-C is a T-lymphocyte-specific immunosuppressant.