ABSTRACT
OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied proinflammatory interleukin (IL) secretion following OK-432 stimulation of total blood, peripheral blood mononuclear cell (PBMC) and purified monocytes in vitro. OK-432 stimulation of purified monocytes gave IL-1beta, IL-1RA, IL-6, IL-12p40 and tumour necrosis factor (TNF)-alpha response. OK-432 stimulation of cells within blood did, however, not yield TNF-alpha secretion. When PBMC or monocytes were cultured in low-attachment wells a decreased IL secretion was observed compared to adherent cells. Inhibition of Syk kinase with piceatannol, only at high, non-specific doses, but not PI3 kinase inhibition with LY294002 or Wortmannin, decreased monocyte IL response to OK-432. This shows that beta(1-3)-integrin receptor function is not necessary for monocyte OK-432-stimulated TNF-alpha secretion. Direct blockage of the beta(2)-integrin (CD18) receptor by anti-CD18 antibody was also unable to prevent the stimulating effects of OK-432 in human monocytes. On the other hand, Syk phosphorylation is elevated upon adherence of monocytes and this is further increased by OK-432 stimulation, as shown by Western blot. The Fc-receptor was also ruled out as a main receptor of the OK-432 monocyte response. In conclusion, TNF-alpha secretion is only found in monocytes removed from blood. This TNF-alpha secretion is not mediated through the beta(1-3)-integrin receptors. OK-432 may act as a target-seeking substance whereby only monocytes adhered, e.g. to a tumour cell, become cytotoxic in part explaining why OK-432 is well suited as a cancer treatment drug.
Subject(s)
Head and Neck Neoplasms/blood , Macrophages/immunology , Monocytes/metabolism , Picibanil/administration & dosage , Tumor Necrosis Factor-alpha/metabolism , Adult , Cell Adhesion/immunology , Cell Differentiation/drug effects , Cells, Cultured , Cytotoxicity, Immunologic/drug effects , Enzyme Inhibitors , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/pathology , Humans , Immunity, Cellular/drug effects , Interferon-gamma/immunology , Interleukins/immunology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/chemistry , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Macrophages/cytology , Male , Middle Aged , Monocytes/cytology , Monocytes/immunology , Picibanil/blood , Picibanil/chemistry , Protein-Tyrosine Kinases/antagonists & inhibitors , Reference Values , Syk KinaseABSTRACT
Bacterial stimulation may serve to control atopic disorders such as atopic dermatitis (AD) through inducement of Th1 cell-mediated immune response. The lipoteichoic acid (LTA)-related molecule (okLTA) from streptococcal preparation, OK-432, has been shown to be a potent Th1 inducer through the action of IL-12. Examination was made of the therapeutic effects of this okLTA injected intra- and/or subcutaneously into AD-like lesions in NC/Nga mice, particularly in the vicinity of the suppressor of cytokine signaling (SOCS) regulatory pathways. Using immunohistochemical staining with IL-4/IL-12p40 and phosphorylated STAT6/p-STAT4 and RT-PCR for IL-4/IL-12p40, STAT6/STAT4 and mRNA expression and in situ hybridization of SOCS3 and 5, evaluation was made of the immunoregulatory effects of this okLTA in the treatment of spontaneous AD-like lesions in NC/Nga mice. Following the injection of okLTA, remarkable improvement in the lesions of NC/Nga mice was noted. In okLTA-treated skin, IL-12p40/p-STAT4 positive cellular infiltration was extensive while IL-4/p-STAT6 positive cell infiltration was seen to diminish considerably, compared to untreated NC mice. SOCS3 in situ expression in okLTA-treated mice was noted to be significantly less compared to untreated NC mice, in which the expression was prominent. SOCS5 in situ expression was rather, though not significantly, strong in okLTA-treated mice. okLTA treatment is clearly shown to induce Th1 cellular response and down-regulate immune response in the Th2 pathway through SOCS3 reduction in AD-like lesions of NC/Nga mice. The present results demonstrate that bacterial wall components such as okLTA should serve as an effective new therapeutic approach for treating AD.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Dermatitis, Atopic/drug therapy , Lipopolysaccharides/pharmacology , Picibanil/pharmacology , STAT6 Transcription Factor/drug effects , Teichoic Acids/pharmacology , Th1 Cells/drug effects , Th2 Cells/drug effects , Animals , Dermatitis, Atopic/pathology , Down-Regulation/drug effects , Immunohistochemistry , In Situ Hybridization , Lipopolysaccharides/chemistry , Mice , Mice, Inbred BALB C , Models, Animal , Monocytes/metabolism , Picibanil/chemistry , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/drug effects , Teichoic Acids/chemistry , Th1 Cells/immunology , Th2 Cells/immunology , Up-Regulation/drug effectsABSTRACT
OK-432 is a Streptococcus-derived immunotherapeutic agent for malignancies. Our group has tried to identify the effective components of OK-432 and has succeeded in isolating a lipoteichoic acid-related preparation designated as OK-PSA, which is a strong inducer of T helper 1 (T(H)1) cells, and elicits an anticancer effect via Toll-like receptor (TLR) 4. Conversely, bacterial DNA with unmethylated CpG motifs can stimulate a T(H)1-type host response via TLR9. The unmethylated CpG DNA contained in OK-432 may play a role in its anticancer effect. In the current study, we investigated the effect of OK-432-derived DNA (OK-DNA) in augmenting the anticancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and MspI revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced TH1-type cytokines such as interferon-gamma, tumor necrosis factor-alpha, interleukin (IL)-12, and IL-18 and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA-stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. The antitumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced compared with that in wild-type mice, whereas the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG DNA in OK-432 functions as an active component in OK-432-induced anticancer immunity via TLR9.
Subject(s)
Antineoplastic Agents/pharmacology , Cytotoxicity, Immunologic/drug effects , Neoplasms, Experimental/drug therapy , Picibanil/chemistry , Picibanil/pharmacology , Toll-Like Receptor 4/immunology , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , CpG Islands , Cytotoxicity, Immunologic/immunology , DNA Methylation , DNA, Bacterial/chemistry , DNA, Bacterial/pharmacology , Humans , Immunotherapy , Mice , Mutation , Neoplasms, Experimental/immunology , Streptococcus pyogenes , Th1 Cells/drug effects , Th1 Cells/immunology , Toll-Like Receptor 4/deficiency , Toll-Like Receptor 4/genetics , Toll-Like Receptor 9/immunologyABSTRACT
BACKGROUND: OK-PSA, a lipoteichoic acid (LTA)-related molecule isolated from a streptococcal agent OK-432, enhances anti-tumor immunity as a potent inducer of Th1-type cytokines. Recently, we obtained the data suggesting that natural killer (NK) cells may play a significant role for OK-PSA-induced cytokine production in vitro. MATERIALS AND METHODS: We conducted the animal experiments using athymic nude mice bearing human salivary adenocarcinoma to examine the role of NK cells in OK-PSA-induced anti-tumor immunity. OK-PSA was peritumorally injected into the mice. Cytokines in the sera were analyzed by ELISA. mRNAs for cytokines were detected by RT-PCR. 51Cr release test was performed to measure killer cell activities. RESULTS: OK-PSA markedly increased the amounts of IFN-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18 that are generally called "Th1-type cytokines" in the sera derived from tumor-bearing nude mice, and also accelerated the killing activities of tumor-infiltrating lymphocytes as well as of draining lymph node cells. Furthermore, OK-PSA administration resulted in significant inhibition of tumor growth, but the effect of OK-PSA was almost completely inhibited by the deletion of NK cells using anti-asialo GM1 antibody. CONCLUSION: These findings strongly suggested that NK cells are closely involved in OK-PSA-mediated anti-tumor immunity.
Subject(s)
Adenocarcinoma/immunology , Adenocarcinoma/therapy , Killer Cells, Natural/immunology , Lipopolysaccharides/pharmacology , Salivary Gland Neoplasms/immunology , Salivary Gland Neoplasms/therapy , Teichoic Acids/pharmacology , Adenocarcinoma/radiotherapy , Animals , Antibodies/immunology , Antibodies/pharmacology , Cell Movement/drug effects , Cell Movement/immunology , Cell Movement/radiation effects , Combined Modality Therapy , Cytokines/biosynthesis , Cytokines/blood , Cytokines/genetics , G(M1) Ganglioside/immunology , Humans , Immunotherapy/methods , Lipopolysaccharides/immunology , Lymphocytes/drug effects , Lymphocytes/immunology , Lymphocytes/radiation effects , Lymphocytes, Tumor-Infiltrating/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Nitric Oxide/biosynthesis , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Picibanil/chemistry , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Salivary Gland Neoplasms/radiotherapy , Teichoic Acids/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Xenograft Model Antitumor AssaysABSTRACT
We isolated the lipoteichoic-acid-related molecule (OK-PSA) from OK-432, a streptococcal preparation, by affinity chromatography on CNBr-activated Sepharose-4B-bound monoclonal antibody TS-2, which neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. We have previously reported that OK-PSA is a potent inducer of Th1-type cytokines in human peripheral blood mononuclear cells in vitro. In this study, we conducted an animal experiment to examine whether OK-PSA exhibits an anti-tumor effect in vivo by acting as a Th1 inducer in syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. It was found that OK-PSA induced Th1-type cytokines [IFN-gamma, tumor necrosis factor-alpha, interleukin (IL)-2, IL-12 and IL-18] in BALB/c mice bearing Meth-A tumor and caused a marked anti-tumor effect. Although it was suggested by an in vitro study. using spleen cells derived from the animals, that IL-18 plays the greatest role in the induction of the Th1-dominant state and tumor cell killing induced by OK-PSA, the in vivo experiments demonstrated that both IL-12 and IL-18 are essential in the anti-tumor effect exhibited by OK-PSA. These findings strongly suggest that OK-PSA is a major effector molecule of OK-432 and may be a useful immunotherapeutic agent, as a potent Th1 inducer, for cancer patients with a Th2-dominant state.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Antineoplastic Agents/therapeutic use , Lipopolysaccharides/therapeutic use , Streptococcus pyogenes/immunology , Teichoic Acids/therapeutic use , Th1 Cells/drug effects , Adjuvants, Immunologic/isolation & purification , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/isolation & purification , Apoptosis , Chromatography, Affinity , Drug Screening Assays, Antitumor , Fas Ligand Protein , Female , Fibrosarcoma/chemically induced , Fibrosarcoma/immunology , Fibrosarcoma/pathology , Fibrosarcoma/therapy , Interleukin-12/antagonists & inhibitors , Interleukin-12/physiology , Interleukin-18/antagonists & inhibitors , Interleukin-18/physiology , Killer Cells, Natural/immunology , Lipopolysaccharides/chemistry , Lipopolysaccharides/pharmacology , Lymphokines/blood , Lymphokines/metabolism , Lymphoma/immunology , Lymphoma/pathology , Lymphoma/therapy , Lymphoma/virology , Membrane Glycoproteins/physiology , Mice , Mice, Inbred BALB C , Moloney murine leukemia virus , Neoplasm Transplantation , Penicillin G/pharmacology , Perforin , Picibanil/chemistry , Pore Forming Cytotoxic Proteins , Spleen/immunology , Spleen/pathology , Streptococcus pyogenes/chemistry , Streptococcus pyogenes/drug effects , Teichoic Acids/chemistry , Teichoic Acids/pharmacology , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/drug effects , Th2 Cells/immunology , Th2 Cells/metabolism , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolism , fas Receptor/physiologyABSTRACT
A lipoteichoic acid-related molecule (OK-PSA) isolated from OK-432, a penicillin-killed Streptococcus pyogenes, is a potent inducer of Th1 cytokines, and elicits anti-cancer effect in tumor-bearing mice. Toll-like receptor (TLR) 4 is a member of the recently identified toll-like receptor family of proteins that has been implicated in lipopolysaccharide-induced cell signaling. In the present study, we have examined the role of TLR4 for OK-PSA-induced Th1-cytokine production and anti-tumor effect by using C3H/HeJ mice in which TLR4 function is impaired. Although OK-PSA strikingly induced Th1 cytokines [interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18] in the splenocytes derived from control animals (C3H/HeN), OK-PSA did not induce the cytokines in the splenocytes from C3H/HeJ. Furthermore, C3H/HeJ-derived splenocytes acquired the responsiveness to OK-PSA stimulation by overexpression of TLR4 gene. Finally, OK-PSA administration significantly inhibited the tumor growth and lung metastasis of syngeneic squamous cell carcinoma cells in C3H/HeN; however, no effect of OK-PSA was observed in C3H/HeJ. These findings strongly suggest that TLR4 signaling is involved in regulating OK-PSA-induced anti-cancer immunity.
Subject(s)
Antineoplastic Agents/pharmacology , Drosophila Proteins , Lipopolysaccharides/pharmacology , Membrane Glycoproteins/deficiency , Penicillins/pharmacology , Picibanil/chemistry , Receptors, Cell Surface/deficiency , Streptococcus pyogenes/chemistry , Teichoic Acids/pharmacology , Animals , Antineoplastic Agents/chemistry , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/pathology , Cell Line , Culture Media , Lipopolysaccharides/chemistry , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C3H , Mice, Knockout , Mutation/genetics , Receptors, Cell Surface/genetics , Spleen/cytology , Spleen/drug effects , Spleen/immunology , Streptococcus pyogenes/drug effects , Teichoic Acids/chemistry , Toll-Like Receptor 4 , Toll-Like Receptors , Transfection , Tumor Cells, CulturedABSTRACT
An immunoglobulin M mouse monoclonal antibody (MAb) to streptococcal preparation OK-432, TS-2, was generated. The TS-2 MAb showed positive reaction with butanol extract of OK-432 and fungal, branched (1-->3)-gamma-glucans (lentinan and schizophyllan) as well as lipoteichoic acids. Moreover, the interferon (IFN)-gamma-inducing activity of OK-432 was neutralized by TS-2 MAb. The affinity column of butanol extract of OK-432 on CNBr-activated Sepharose 4B-bound TS-2 antibody was prepared and the fractions containing IFN-gamma-inducing activity were eluted. The polysaccharide sample carrying the IFN-gamma-inducing activity with a molecular weight of 700,000 was destroyed by treatment with acid or sodium metaperiodate, but was stable to treatment with heat, alkali, pronase, or neuraminidase. Survival time of human salivary adenocarcinoma-bearing animals given a combination of the polysaccharide sample purified from butanol extract of OK-432 and TS-2 MAb was significantly shorter compared with that of the tumor-bearing animals given only the purified polysaccharide sample of OK-432. Moreover, a high level of effector cell activity in natural killer (NK) and lymphokine-activated killer (LAK) assays and significant increase of IFN-gamma-positive cells or asialo-GM1-positive cells were detected in the spleen cells from the animals given the polysaccharide sample purified from butanol extract of OK-432. These findings indicate that the polysaccharide sample purified by the affinity chromatography of butanol extract of OK-432 on CNBr-activated Sepharose 4B-bound TS-2 MAb carries the IFN-gamma-inducing activity of OK-432 and marked antitumor activity.
