Staphylococcal superantigen-like protein 3 triggers murine mast cell adhesion by binding to CD43 and augments mast cell activation.

Staphylococcus aureus is a noteworthy pathogen in allergic diseases, as four staphylococcal exotoxins activate mast cells, a significant contributor to inflammation, in an IgE-independent manner. Although the adhesion of mast cells is an essential process for their immune responses, only a small number of exotoxins have been reported to affect the process. Here, we demonstrated that staphylococcal superantigen-like (SSL) 3, previously identified as a toll-like receptor 2 agonist, induced the adhesion of murine bone marrow-derived mast cells to culture substratum. SSL3-induced adhesion was mediated by fibronectin in an Arg-Gly-Asp (RGD) sequence-dependent manner, suggesting the integrins were involved in the process. Additionally, SSL3 was found to bind to an anti-adhesive surface protein CD43. SSL3 induced the adhesion of HEK293 cells expressing exogenous CD43, suggesting that CD43 is the target molecule for adhesion induced by SSL3. Evaluation of SSL3-derived mutants showed that the C-terminal region (253-326), specifically T285 and H307, are necessary to induce adhesion. SSL3 augmented the IL-13 production of mast cells in response to immunocomplex and SSL12. These findings reveal a novel function of SSL3, triggering cell adhesion and enhancing mast cell activation. This study would clarify the correlation between S. aureus and allergic diseases such as atopic dermatitis.

Interleukin-3-dependent potentiation of IgE responsiveness in mouse basophils.

Basophils produce interleukins (IL)-4 in response to various stimuli and may contribute to type 2 immune responses to various infections and allergens. We found that resting basophils freshly isolated from mice produce IL-4 in response to IL-3 but not to high-affinity Fc receptor (FcεRI) cross-linking (CL), yet both required the immunoreceptor tyrosine-based activation motif (ITAM) containing adaptor Fc receptor γ-chain (FcRγ), while basophils activated in vitro by IL-3 become responsive to FcεRI CL. Acquisition of responsiveness to FcεRI CL occurred upon infection with Trichinella spiralis or administration of superantigen. Because cultured basophils return to a quiescent state upon starvation with IL-3 with surface FcεRI levels unchanged, this acquisition is reversible and probably reflects intracellular events requiring protein synthesis. Interestingly, similar activation-associated acquisition was observed for responsiveness to other stimuli, including CD200R3 CL, which is known to signal via DAP-12, and the allergen protease papain. This acquisition of responsiveness to FcεRI CL was inhibited by Jak inhibitor. Thus, the IL-3 signal bifurcates downstream of Jak, into two distinct pathway, one leading to IL-4 production and the other to render basophils competent to respond to stimuli dependent on ITAM-containing adaptors DAP12 and FcRγ for IL-4 production.

Staphylococcal γ-hemolysins induce IL-4 production in murine basophils.

Basophils are known to produce a large amount of IL-4 in response to stimuli and play a role in the initiation and propagation of type 2 inflammations. S. aureus secretes a series of pore-forming toxins: α-hemolysin, γ-hemolysins, and leukocidins. In this study, we examined the effects of α-hemolysin, γ-hemolysins (HlgAB and HlgCB), and leukocidins (LukAB, LukED, and Panton-Valentine leukocidin) on the function of basophils. All pore-forming toxins except for Panton-Valentine leukocidin bound to murine bone marrow-derived basophils (BMBs). HlgAB and LukED but not other toxins evoked the leakage of lactate dehydrogenase from BMBs at the concentration of 30 µg/ml γ-hemolysins, HlgAB and HlgCB, induced the secretion of IL-4 in BMBs at concentrations above 3.3 µg/ml. LukAB did not induce, and Hla and LukED induced only a small amount of IL-4. HlgBΔstem, the 5 amino acids deletion mutant of HlgB in the stem region, diminished IL-4 secretion by HlgAB and HlgCB in BMBs. These results suggest that the cell damage and the induction of IL-4 in basophils by HlgAB require pore formation. The induction of IL-4 by γ-hemolysins was also observed in fleshly isolated murine basophils. These results demonstrate a novel function of γ-hemolysins, the induction of IL-4 in basophils, in an IgE-independent manner.

Identification of responsible amino acid residues in staphylococcal superantigen-like 12 for the activation of mast cells.

Staphylococcal superantigen-like 12 (SSL12) is reported to evoke the degranulation in murine mast cells. The allelic variant of SSL12 in the genome of reference strain NCTC8325 induced the degranulation of murine mast cells, that of MRSA252 strain did not, nevertheless relatively high sequence similarity (82%). To identify responsible amino acid residues of SSL12 for mast cell activation, we created a series of domain swap mutants and amino acid substitution mutants between the active and inactive variants. The mutants that harbored oligonucleotide/oligosaccharide binding (OB)-fold domain of the active variant activated mast cells. The replacement at position 56 (L56F) in the OB-fold domain diminished the mast cell stimulatory activity, and the combinatorial substitutions L56F/K92E, L56F/D95S, and L56F/S100V abolished the stimulatory activities of the mutant that harbored OB-fold domain of the active variant and the intact active variant. These indicate that the responsive elements of SSL12 for mast cell activation are in the OB-fold of SSL12, and L56 would be an essential amino acid residue for the activation of mast cells. The findings would contribute to the understanding of the molecular mechanism of SSL12 for mast cell activation and the development of toxoids preventing allergic inflammations associated with Staphylococcus aureus.

Comparative Study of the Susceptibility to Oxidative Stress between Two Types of Mycobacterium bovis BCG Tokyo 172.

Genomic analysis revealed that the vaccine seed lot of Mycobacterium bovis bacillus Calmette-Guérin (BCG) Tokyo 172 contains two subclones (types I and II), but their phenotypic differences have not been elucidated. In this study, we compared the susceptibility of bacilli types I and II to oxidative stress in vitro and within host cells. Notably, the subclones displayed similar superoxide dismutase activity; however, foam height in the catalase test and lysate catalase/peroxidase activity were higher for type I bacilli than for type II bacilli. Additionally, type I bacilli were less susceptible to hydrogen peroxide (H2O2) than type II bacilli. After exposure to H2O2, antioxidative stress response genes katG, ahpC, sodA, and trxA were more strongly induced in type I bacilli than in type II bacilli. Further, we investigated cell survival in macrophages. Fewer type II bacilli were recovered than type I bacilli. However, in the presence of apocynin, a specific inhibitor of NADPH oxidase, type II recovery was greater than that of type I. The production of interleukin 1ß (IL-1ß), IL-12 p40, and tumor necrosis factor alpha (TNF-α) was higher in type I bacillus-infected macrophages than in type II bacillus-infected macrophages. The proportions of type I and type II bacilli in vaccine lots over 3 years (100 lots) were 97.6% ± 1.5% and 2.4% ± 1.5%, respectively. The study results illustrated that type I bacilli are more resistant to oxidative stress than type II bacilli. Overall, these findings provide important information in terms of the quality control and safety of BCG Tokyo 172 vaccine.IMPORTANCE This study revealed the difference of in vivo and in vitro antioxidative stress properties of BCG Tokyo 172 types I and II as one of the bacteriological characteristics. In particular, the bacilli exhibited differences in catalase/peroxidase activity, which could explain their different protective effects against infection. The differences correlated with survival in the host cell and the production of proinflammatory cytokines to protect against infection by Mycobacterium tuberculosis The proportion of bacilli types I and II in all commercial lots of BCG Tokyo 172 over 3 years (100 lots) was constant. The findings also highlighted the importance of analyzing their content for quality control during vaccine production.

Staphylococcal superantigen-like 12 induces the production of interleukin 4 in murine basophils.

S. aureus is associated with atopic dermatitis (AD). Several staphylococcal products including cell wall components, protease, and exotoxins, are thought to be involved in allergic inflammation of AD via activating immune cells such as T cells and mast cells. None of the staphylococcal exotoxins has been reported to activate a primary IL-4 inducer, basophils, that are known to produce large amounts of IL-4 in response to allergens as well as IgE-independent stimuli such as mites and helminth proteases. In this study, we investigated the ability of staphylococcal superantigen-like (SSL) family to activate basophils. SSL12, reported its activity to activate mast cells, induced the production of IL-4 in bone marrow derived basophils. SSL12 also evoked the release of IL-4 in freshly isolated murine basophils in bone marrow cells, as the depletion of basophils by basophils-specific antibodies against high-affinity IgE receptor and CD49b diminished the responsiveness of bone marrow cells for SSL12. These results propose the novel immune regulatory activity of SSL12 by inducing IL-4 in basophils, that contributes to the development of allergic inflammation disorders and the immune evasion of the cocci.

Selective Cytotoxicity of Staphylococcal α-Hemolysin (α-Toxin) against Human Leukocyte Populations.

Staphylococcus aureus produces a variety of exoproteins that interfere with host immune systems. We attempted to purify cytotoxins against human leukocytic cells from the culture supernatant of S. aureus by a combination of ammonium sulfate precipitation, ion-exchange chromatography on a CM-cellulose column and HPLC on a Mono S 5/50 column. A major protein possessing cytotoxicity to HL60 human promyelocytic leukemia cells was purified, and the protein was identified as α-hemolysin (Hla, α-toxin) based on its molecular weight (34 kDa) and N-terminal amino acid sequence. Flow cytometric analysis suggested differential cytotoxicity of Hla against different human peripheral blood leukocyte populations. After cell fractionation with density-gradient centrifugation, we found that peripheral blood mononuclear cells (PBMCs) were more susceptible to Hla than polymorphonuclear leukocytes. Moreover, cell surface marker analysis suggested that Hla exhibited slightly higher cytotoxicity against CD14-positive PBMCs (mainly monocytes) than CD3- or CD19-positive cells (T or B lymphocytes). From these results, we conclude that human leukocytes have different susceptibility to Hla depending on their cell lineages, and thereby the toxin may modulate the host immune response.

Staphylococcal superantigen-like 12 activates murine bone marrow derived mast cells.

Staphylococcal superantigen-like (SSL) protein is a family of exotoxins that consists of 14 SSLs, and the roles of several SSLs in immune evasion of the cocci have been revealed. However little is known whether they act as immune activators and are involved in inflammatory disorders such as atopic dermatitis. In this study we examined whether SSLs activate mast cells, the key player of local inflammation. SSL12 evoked the release of a granule enzyme ß-hexosaminidase from bone marrow derived mast cells (BMMCs) in the absence of IgE. The release of the granule enzyme caused by SSL12 was not accompanied with the leakage of a cytosolic enzyme lactate dehydrogenase (LDH), unlike staphylococcal δ-toxin that was reported to induce both the release of ß-hexosaminidase and the leakage of LDH from the cells, suggesting that SSL12 evokes the degranulation of mast cells without cell membrane damage. Furthermore SSL12 induced IL-6 and IL-13 in both mRNA and protein levels indicating that SSL12 induces de novo synthesis of the cytokines. Evans blue extravasation was elevated by the intradermal injection of SSL12, suggesting that SSL12 is also able to evoke local inflammation in vivo. These findings indicate the novel mast cell activating activity of SSLs, and SSL12 is likely an important factor in both initiation phase and effector phase of allergic and immune responses.

Staphylococcal α-hemolysin does not induce cell damage in murine mast cells but it augments the degranulation induced by FcεRI cross-linking and ionomycin.

Staphylococcal α-hemolysin (Hla) is a principal small ß-barrel pore forming toxin. It targets a variety of mammalian cells including immune cells; however little is known about its effects on mast cells. In this study, we examined whether Hla affects the degranulation of mast cells. Although Hla bound to the surface of bone marrow-derived mast cells (BMMCs) and formed SDS-stable oligomers on the cells, Hla alone induced neither cytotoxicity nor obvious release of a granule enzyme, ß-hexosaminidase. However, Hla more than doubled the releases of ß-hexosaminidase from BMMCs induced by FcεRI cross-linking or treatment with ionomycin. The augmentation of the enzyme release by rHla was impaired in the presence of 130 mM of extracellular KCl. The mutants of Hla that lacked pore-formation did not augment the release of the enzyme. These findings demonstrate that Hla is able to enhance the degranulation of mast cells induced by FcεRI cross-linking and ionomycin, although it alone does not induce the degranulation, and the pore-formation of Hla followed by potassium efflux is involved in the augmentation. These findings propose a previously unrecognized role for Hla in S. aureus-associated allergic and inflammatory processes via augmentation of mast cell responses.

5-Hydroxy-2-methylpyridine Isolated from Cigarette Smoke Condensate Aggravates Collagen-Induced Arthritis in Mice.

The risk of rheumatoid arthritis (RA) is linked to environmental and genetic factors. Cigarette smoking is an established environmental risk factor for the disease that contributes to its development and severity. Previously, we found that cigarette smoke condensate (CSC), both mainstream and sidestream, aggravates collagen type II-induced arthritis (CIA), which was observed following either intraperitoneal inoculation or nasal exposure. In the present study, we aimed to identify the compound in CSC, which aggravates CIA. By sequential fractionation and analysis, extraction with water/ether in different pH values, silica gel column chromatography, TLC, octadecyl silica (ODS) HPLC, GC/MS, and NMR, the active compound was identified as 5-hydroxy-2-methylpyridine (5H2MP). Its isomer 2-hydroxy-3-methylpyridine, but not 3-hydroxy-2-methylpyridine, was also active. 5H2MP was not mutagenic, and did not exhibit aryl hydrocarbon receptor-dependent activity. Our data help clarify the mechanism underlying the pathogenic effects of cigarette smoking on RA.

Identification of matrix metalloproteinase 9-interacting sequences in staphylococcal superantigen-like protein 5.

Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, ß1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4ß9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved ß1-3 but not α4ß9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted ß1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored ß1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the ß1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.

Role of sialic acid-containing glycans of matrix metalloproteinase-9 (MMP-9) in the interaction between MMP-9 and staphylococcal superantigen-like protein 5.

Staphylococcal superantigen-like proteins (SSL) show no superantigenic activity but have recently been considered to act as immune suppressors. It was previously reported that SSL5 bound to P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase (MMP)-9, leading to inhibition of leukocyte adhesion and invasion. These interactions were suggested to depend on sialic acid-containing glycans of MMP-9, but the roles of sialic acids in the interaction between SSL5 and MMP-9 are still controversial. In the present study, we prepared recombinant glutathione S-transferase-tagged SSL5 (GST-SSL5) and analyzed its binding capacity to MMP-9 by pull-down assay after various modifications of its carbohydrate moieties. We observed that GST-SSL5 specifically bound to MMP-9 from a human monocytic leukemia cell line (THP-1 cells) and inhibited its enzymatic activity in a concentration-dependent manner. After MMP-9 was treated with neuraminidase, its binding activity towards GST-SSL5 was markedly decreased. Furthermore, recombinant MMP-9 produced by sialic acid-deficient Lec2 mutant cells showed much lower affinity for SSL5 than that produced by wild-type CHO-K1 cells. Treatment of MMP-9 with PNGase F to remove N-glycan resulted in no significant change in the GST-SSL5/MMP-9 interaction. In contrast, the binding of GST-SSL5 to MMP-9 secreted from THP-1 cells cultured in the presence of an inhibitor for the biosynthesis of O-glycan (benzyl-GalNAc) was weaker than the binding of GST-SSL5 to MMP-9 secreted from untreated cells. These results strongly suggest the importance of the sialic acid-containing O-glycans of MMP-9 for the interaction of MMP-9 with GST-SSL5.

Identification of the blood coagulation factor interacting sequences in staphylococcal superantigen-like protein 10.

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins of Staphylococcus aureus. We have shown that SSL10 binds to vitamin K-dependent coagulation factors and inhibits blood coagulation induced by recalcification of citrated plasma. SSL10 was revealed to bind to coagulation factors via their γ-carboxyglutamic acid (Gla) domain. In this study we attempted to identify the responsible sequence of SSL10 for the interaction with coagulation factors. We prepared a series of domain swap mutants between SSL10 and its paralog SSL7 that does not interact with coagulation factors, and examined their binding activity to immobilized prothrombin using ELISA-like binding assay. The domain swap mutants that contained SSL10ß1-ß3 (23MEMKN ISALK HGKNN LRFKF RGIKI QVL60) bound to immobilized prothrombin, and mutants that contained SSL10ß10-ß12 (174SFYNL DLRSK LKFKY MGEVI ESKQI KDIEV NLK207) also retained the binding activity. On the other hand, mutants that lacked these two regions did not bind to prothrombin. These sequences, each alone, bound to prothrombin as 33 amino acid length polypeptides. These results suggest that SSL10 has two responsible sequences for the binding to prothrombin. These prothrombin-binding peptides would contribute to the development of new anticoagulants.

Early-shared Mycobacterium bovis bacillus Calmette-Guérin sub-strains induce Th1 cytokine production in vivo.

Interleukin-12 is one of the cytokines that induce acquired immunity by progressing the differentiation of T cells. When antigens are presented by APCs, including macrophages and DCs, T cells are activated and produce the Th1 cytokines IL-2 and IFN-γ. We have previously reported greater IL-12 production from macrophages infected with early-shared BCG sub-strains (ex. BCG-Japan, -Sweden) than from those infected with late-shared BCG (ex. BCG-Pasteur and -Connaught) . In this study, we investigated the Th1 cytokine-inducing activity of splenocytes co-cultured with BCG-infected DCs. Early-shared BCG-infected DCs produced IL-12 and TNF-α⋅ Furthermore, when they were co-cultured with purified protein derivative-stimulated DCs, the splenocytes of mice immunized with BCG-Tokyo/Japan produced more Th1 cytokine than did those of mice immunized with BCG-Connaught. In conclusion, early-shared BCG sub-strains more strongly induce Th1 cytokine production in vivo. This study provides basic information to inform the selection of candidates for primary vaccination.

Development of liposomal nanoconstructs targeting P-selectin (CD62P)-expressing cells by using a sulfated derivative of sialic acid.

PURPOSE: NMSO3, a sulfated derivative of sialic acid, is a specific inhibitor for P-selectin (CD62P)-mediated cell adhesion. We attempted to apply liposomes modified with NMSO3 for selective targeting of activated platelets. METHODS: The binding of fluorescently labeled NMSO3-containing liposomes (NMSO3-liposomes) to CHO cells expressing P-selectin (CHO-P cells) and activated platelets were examined. The distribution of NMSO3-liposomes incorporated into the cells was observed by fluorescence microscopy. RESULTS: The binding assay revealed that NMSO3-liposomes specifically bound to immobilized P-selectin and CHO-P cells in a dose-dependent manner. The binding of NMSO3-liposomes to CHO-P cells was much stronger than that to the parental CHO-K1 cells. Fluorescence microscopic observation showed that NMSO3-liposomes were incorporated into CHO-P cells after the binding and distributed throughout the cytoplasm of the cell. NMSO3-liposomes bound more strongly to thrombin-activated platelets than to resting platelets, as assessed by flow cytometry. CONCLUSIONS: These results suggest that NMSO3-liposomes can be applied for selective drug delivery to activated platelets.

Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

Reactivation of immune responses against Mycobacterium tuberculosis by boosting with the CpG oligomer in aged mice primarily vaccinated with Mycobacterium bovis BCG.

BACKGROUND: Mycobacterium bovis bacillus Calmette Guérin (BCG) vaccine, which has been inoculated to more than one billion people world-wide, has significant effect in preventing tuberculous meningitis and miliary tuberculosis (TB) in neonate and early childhood. However, BCG fails to adequately protect against pulmonary TB and reactivation of latent infections in adults. To overcome this problem, adequate booster is urgently desired in adult who received prior BCG vaccination, and appropriate animal models that substitute human cases would be highly valuable for further experimentation. FINDINGS: The booster effect of the synthesized CpG oligomer (Oligo-B) on aged mice which had been primarily vaccinated with BCG at the age of 4-week old. The specific Th1 type reaction, production of interferon-γ, in response to TB antigens, purified protein derivatives (PPD) and protection against challenge with Mycobacterium tuberculosis (MTB) H37Rv decreased with increasing age and were not observed in 89-week old mice. In order to rejuvenate the Th1 type response against PPD and protection activity against MTB infection, Oligo-B, which is known to augment Th1 responses, was administered as a booster to 81-90-week old mice (late 50's in human equivalent) vaccinated with BCG at 4-week old. The boosting with Oligo-B increased the number of CD4+ CD44high CD62Lhigh, central memory type T cell. Furthermore, the Oligo-B boosting rejuvenated the ability of mice to protect against infection with MTB H37Rv. CONCLUSIONS: Th1-adjuvant CpG oligo DNA, such as Oligo-B, may be a promising booster when coupled with BCG priming.

Staphylococcal superantigen-like protein 8 (SSL8) binds to tenascin C and inhibits tenascin C-fibronectin interaction and cell motility of keratinocytes.

Staphylococcal superantigen-like protein (SSL), a family of exotoxins composed of 14 SSLs, exhibits no superantigenic activity despite of its structural similarity with superantigens. Several SSLs have been revealed to bind to host immune molecules such as IgA, IgG, complement and cell surface molecules expressed on immune cells, but the physiological function of SSL family has not been fully identified. In this study we attempted to isolate host target proteins of SSLs from human breast milk using SSLs-conjugated Sepharose. SSL8-conjugated Sepharose specifically recovered tenascin C (TNC), a multimodular and multifunctional extracellular matrix protein. Pull down analysis using SSL8-conjugated Sepharose and recombinant truncated fragments of TNC revealed that SSL8 interacts with fibronectin (FN) type III repeats 1-5 of TNC. The interaction of TNC with immobilized FN was attenuated, the scratch wound closure by HaCaT human keratinocytes was delayed and the inhibition of cell spreading on FN by TNC was recovered in the presence of SSL8. These findings suggest that SSL8 binds to TNC, thereby inhibits the TNC-FN interaction and motility of keratinocytes. The present study added a novel role of SSL family protein as an interrupting molecule against the function of extracellular matrix.

Cigarette smoke condensate extracts induce IL-1-beta production from rheumatoid arthritis patient-derived synoviocytes, but not osteoarthritis patient-derived synoviocytes, through aryl hydrocarbon receptor-dependent NF-kappa-B activation and novel NF-kappa-B sites.

Cigarette smoking is a major established environmental risk factor for rheumatoid arthritis (RA), and synoviocyte-derived proinflammatory cytokines are implicated in the pathogenesis of RA. We have reported that aryl hydrocarbon or cigarette smoke condensate (CSC) is able to upregulate the production of proinflammatory cytokines from an RA patient-derived synovial fibroblast cell line MH7A. In this study, we compared the effect of CSC on induction of interleukin-1ß (IL-1ß) from RA or osteoarthritis (OA) patient-derived synovial fibroblasts, and studied the mechanism of the effect of CSC. CSC induced IL-1ß mRNA from RA patient-derived synoviocytes and MH7A, but not from OA patient-derived synoviocytes. CSC induced the mRNA and both precursor and mature forms of IL-1ß, and caspase-1 activity in MH7A. The mechanism of CSC-induced IL-1ß mRNA expression was investigated in MH7A. Reporter gene analyses and promoter pull-down assay indicated that 3 novel NF-κB sites at -3771 to -3762 bp, -3105 to -3096 bp, and -2787 to -2778 bp in the promoter region of the IL-1ß gene, especially the far distal NF-κB site and NF-κB activation, are critical for the gene activation by CSC. CSC-induced NF-κB activation, IL-1ß promoter activity, IL-1ß mRNA upregulation, and CYP1A1 mRNA induction were all inhibited by an aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone. These results indicate that CSC induced IL-1ß production from RA patient-derived synoviocytes, but not OA patient-derived synoviocytes, through AhR-dependent NF-κB activation and novel NF-κB sites.

Cytokine secretion from human monocytes potentiated by P-selectin-mediated cell adhesion.

BACKGROUND/AIM: P-selectin is a carbohydrate-recognizing cell adhesion molecule expressed on activated platelets and endothelial cells. It plays a crucial role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. Cell adhesion mediated by P-selectin induces leukocyte activation, such as the generation of reactive oxygen species and the expression of blood coagulation factors. We assessed how P-selectin-mediated cell adhesion affects cytokine secretion from monocytes. METHODS: Human peripheral blood monocytes were cultured in a plate that had been coated with P-selectin purified from human platelets, and cytokines released in the culture supernatant from monocytes were determined by ELISA. RESULTS: The secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, IL-12 and macrophage inflammatory protein-1ß increased 3- to 10-fold in response to P-selectin compared with unstimulated monocytes. We next examined the effects of cytokine treatment of monocytes on their susceptibility to P-selectin. The secretion of TNF-α from monocytes in response to P-selectin was increased when monocytes were preincubated with granulocyte/macrophage colony-stimulating factor, monocyte chemotactic protein-1 or interferon-γ (IFN-γ); IFN-γ was the most effective in potentiating TNF-α secretion from monocytes. CONCLUSION: These results suggest that the interaction of monocytes with P-selectin plays an important role not only in their trafficking but also in the regulation of cytokine production by these cells.