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The transcriptional regulator IκB-ζ is important for the control of apoptosis in keratinocytes. Thus, IκB-ζ-deficient mice develop autoimmune diseases, such as Sjögren’s syndrome. However, T cells also play a pivotal role in Sjögren’s syndrome. To study the role of IκB-ζ in T cells, we generated T cell-specific, IκB-ζ-deficient mice. We observed increased numbers of peripheral effector/memory CD4+ cells and IFN-γ-producing CD4+ cells in 3-week-old mice. We found that IκB-ζ can be up-regulated by TGF-β1 in naϊve CD4+ T cells and that it negatively regulates IFN-γ expression. In addition, we generated Treg-specific, IκB-ζ deficient mice and found that IκB-ζ is dispensable for the plasticity and stability of Tregs. However, Tregs from T cell-specific, IκB-ζ-deficient mice have reduced immunoregulatory function. Thus, our data reveal a previously unappreciated role for IκB-ζ in IFN-γ production in T cells and the immunoregulatory function of Tregs.

The immune system maintains homeostasis by recognizing and responding to cell death caused by various stresses. The immune response is considered to be elicited by ‘danger signals’ released from necrotic cells. However, the identity of the danger signals remains elusive. In this study, we focused on the expression of chemokines by macrophages stimulated with necrotic cells. In mouse bone-marrow-derived macrophages, the chemokine monocyte chemoattractant protein (MCP)-3 was induced at both the mRNA and protein levels in response to heat-killed murine cells. The induction of MCP-3 was also observed in MyD88-deficient macrophages, indicating that Toll-like receptors and the IL-1 receptor are not involved in this response. Consistent with this observation, the activation of NF-κB was not detected in RAW264.7 macrophages stimulated with necrotic cells. Treatments with proteinase K, DNaseI or RNaseA did not affect the ‘stimulating activity’ of necrotic cells. In contrast, treatment with apyrase, which removes phosphates from nucleoside tri- and di-phosphates, abolished the inducing activity. Purified UDP at 30 µM concentration elicited similar induction of MCP-3 in RAW264.7 macrophages. Small interfering RNA-mediated knock-down of the UDP receptor P2Y6 in RAW264.7 cells significantly reduced the induction of MCP-3 in response to necrotic cells, but not its induction by lipopolysaccharide. Furthermore, ectopic expression of the P2Y6 receptor in HEK293 cells conferred responsiveness to necrotic cells. These results suggest that UDP released by necrotic cells plays a critical role as an endogenous danger signal and that P2Y6 is required for the induction of MCP-3 in response to necrotic cells.

IκB-ζ is a nuclear IκB protein robustly induced in macrophages and fibroblasts upon TLR or IL-1R stimulation. IκB-ζ associates with NF-κB in the cell nucleus and is essential for the induction of a subset of secondary response genes represented by IL-6. Here, we analyzed induction of IκB-ζ in mouse B cells and found that IκB-ζ is induced by BCR or TLR stimulation. Similar to TLR stimulation, BCR stimulation elicited NF-κB-mediated transcriptional activation and mRNA stabilization of IκB-ζ via a cis-element in IκB-ζ mRNA. Proteasome inhibitors inhibited transcriptional activation but not post-transcriptional activation, indicating independency of the two signals. Co-stimulation of the BCR and TLR9 or TLR7, but not TLR2/1, synergistically induced IκB-ζ. Co-engagement of inhibitory Fcγ receptor suppressed BCR-mediated IκB-ζ expression but not that induced by TLR stimulation alone or co-stimulation of TLR and the BCR. The PI3K inhibitor LY294002 inhibited BCR-mediated, but not TLR-mediated, induction of IκB-ζ, consistent with the role of PI3K in BCR signaling and its suppression by FcγR. Analysis of IκB-ζ-deficient B cells demonstrated that IκB-ζ was essential upon stimulation of BCR or TLR for the expression of several genes including IL-10 and CTLA4. IκB-ζ-deficient B cells exhibited impaired proliferation and enhanced up-regulation of CD86 following stimulation of TLR9, but not the BCR, indicating critical roles for IκB-ζ in TLR signaling in B cells. Strict regulatory mechanisms for the induction of IκB-ζ via multiple pathways and its essential function upon stimulation indicate that IκB-ζ plays an important role in B cells.

Background: NF-κB regulates transcription via binding to DNA and interactions with cofactors. Results: NF-κB binding to DNA and cytosolic IκBs is conserved, whereas binding to nuclear IκBs has evolved. Conclusion: There is distinct evolutionary pressure on two NF-κB/IκB binding interfaces. Significance: The results provide inroads into IκB-specific modulation of NF-κB activity., NF-κB is a key regulator of immune gene expression in metazoans. It is currently unclear what changes occurred in NF-κB during animal evolution and what features remained conserved. To address this question, we compared the biochemical and functional properties of NF-κB proteins derived from human and the starlet sea anemone (Nematostella vectensis) in 1) a high-throughput assay of in vitro preferences for DNA sequences, 2) ChIP analysis of in vivo recruitment to the promoters of target genes, 3) a LUMIER-assisted examination of interactions with cofactors, and 4) a transactivation assay. We observed a remarkable evolutionary conservation of the DNA binding preferences of the animal NF-κB orthologs. We also show that NF-κB dimerization properties, nuclear localization signals, and binding to cytosolic IκBs are conserved. Surprisingly, the Bcl3-type nuclear IκB proteins functionally pair up only with NF-κB derived from their own species. The basis of the differential NF-κB recognition by IκB subfamilies is discussed.

Tumor cell-derived factors, such as interleukin 10 (IL-10), polarize macrophages toward a regulatory M2 phenotype, characterized by the expression of anti-inflammatory cytokines and protumorigenic mediators. Here we explored molecular mechanisms allowing IL-10 to upregulate the protumorigenic protein NGAL in primary human macrophages. Reporter assays of full-length or deletion constructs of the NGAL promoter provided evidence that NGAL production is STAT3 dependent, activated downstream of the IL-10-Janus kinase (Jak) axis, as well as being C/EBPβ dependent. The involvement of STAT3 and C/EBPβ was shown by chromatin immunoprecipitation (ChIP) and ChIP-Western analysis, as well as decoy oligonucleotides scavenging both STAT3 and C/EBPβ in human macrophages. Furthermore, the production of NGAL in macrophages in response to IL-10 induces cellular growth and proliferation of MCF-7 breast cancer cells. We conclude that both STAT3 and C/EBPβ are needed to elicit IL-10-mediated NGAL expression in primary human macrophages. Macrophage-secreted NGAL shapes the protumorigenic macrophage phenotype to promote growth of MCF-7 breast cancer cells. Our data point to a macrophage-dependent IL-10-STAT3-NGAL axis that might contribute to tumor progression. © 2012, American Society for Microbiology., The work was supported by grants from Deutsche Forschungsgemeinschaft (BR999, ECCPS), Sander Foundation (2007.070.2), and Deutsche Krebshilfe (109599). M.J. was supported by a grant from the Fritz-Thyssen-Stiftung and from the Medical Faculty, Goethe University Frankfurt. J.M. was supported by the Deutscher Akademischer Austauschdienst (DAAD).

IκB-ζ, an essential inflammatory regulator, is specifically induced by Toll-like receptor ligands or interleukin (IL)-1β by post-transcriptional activation mediated via a 165-nucleotide element in IκB-ζ mRNA. Here, we analyzed the Toll-like receptor–IL-1 receptor signaling components involved in the post-transcriptional regulation of IκB-ζ with mutated estrogen receptor [ER(T2)] fusion proteins. Upon 4-hydroxytamoxifen treatment, the ER(T2) fusion proteins with IL-1 receptor-associated kinase (IRAK)1 and IRAK4 elicited specific activation of a reporter gene for the post-transcriptional regulation of IκB-ζ. The tumor necrosis factor receptor-associated factor (TRAF)6–ER(T2) protein activated nuclear factor-κB, but not post-transcriptional regulation, indicating that activation of IRAK1/4, but not of TRAF6, is sufficient to activate the 165-nucleotide element-mediated post-transcriptional mechanism. Interestingly, the post-transcriptional mechanism was not activated in TRAF6-deficient cells, indicating an essential role for TRAF6. Thus, the signaling pathway leading to nuclear factor-κB activation and the post-transcriptional activation bifurcates at IRAK1, suggesting a new pathway activated by IRAK1.

Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-(L)) and an alternatively initiated or spliced (Bcap-(S)) mRNA, and little is known about the differential functions of the BCAP-(L) and BCAP-(S) proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-(L) enhanced IL-6 and IL-10 production but not TNF-α production in TLR ligand-stimulated macrophages. We propose that BCAP-(L) (but not BCAP-(S)) is a negative regulator of the TLR-mediated host defense system in macrophages.

Horseshoe crab haemolymph contains a single type of cells, granular haemocytes, which are extremely sensitive to bacterial lipopolysaccharides (LPS) and lead to haemolymph coagulation. Sadaaki Iwanaga isolated protease zymogens from the haemocytes and reconstituted LPS and beta-1,3-d-glucans-mediated haemolymph coagulation. This led to the first discovery of a proteolytic cascade triggered by pathogen-associated molecular patterns, an important milestone for studies on invertebrate innate immunity. Moreover, he separated components derived from haemocyte granules and haemolymph plasma, and consequently identified unique defense molecules, such as lectins, serpins, cystatins, antimicrobial substances and substrates for transglutaminase. Through steady and persistent studies on the horseshoe crab host defense system, he made great progress in the field. Now we know that LPS-induced haemocyte exocytosis leads not only to coagulation but also activates a sophisticated immune response network that coordinately induces pathogen recognition, elimination and wound healing.

Expression of many inflammatory genes is induced through activation of the transcription factor NF-kappaB. In contrast to the advanced understanding of cytoplasmic control of NF-kappaB activation, its regulation in the nucleus has not been fully understood despite its importance in selective gene expression. We previously identified an inducible nuclear protein, IkappaB-zeta, and demonstrated that this molecule is indispensable for the expression of a group of NF-kappaB-regulated genes. In this study, we established a unique gene induction system, in which IkappaB-zeta is expressed independently of inflammatory stimuli, to specifically investigate the molecular basis underlying IkappaB-zeta-mediated gene activation. We show that in the presence of IkappaB-zeta other primary response genes are dispensable for the expression of the target secondary response genes. ChIP analyses revealed that IkappaB-zeta is required for stimulus-induced recruitment of NF-kappaB onto the target promoter in a gene-specific manner. Surprisingly, IkappaB-zeta is also necessary for the gene-selective promoter recruitment of another inflammatory transcription factor, C/EBPbeta, and the chromatin remodeling factor Brg1. We propose a new gene regulatory mechanism underlying the selective expression of inflammatory genes.

IkappaB-zeta [inhibitor of NF-kappaB (nuclear factor kappaB) zeta] is a nuclear protein that is induced upon stimulation of TLRs (Toll-like receptors) and IL (interleukin)-1 receptor. IkappaB-zeta harbours C-terminal ankyrin repeats that interact with NF-kappaB. Our recent studies have shown that, upon stimulation, IkappaB-zeta is essential for the induction of a subset of inflammatory genes, represented by IL-6, whereas it inhibits the expression of TNF (tumour necrosis factor)-alpha. In the present study, we investigated mechanisms that determine the different functions of IkappaB-zeta. We found that co-expression of IkappaB-zeta and the NF-kappaB subunits synergistically activates transcription of the hBD-2 (human beta-defensin 2) and NGAL (neutrophil gelatinase-associated lipocalin) genes, whereas it inhibits transcription of E-selectin. Reporter analyses indicated that, in addition to an NF-kappaB-binding site, a flanking C/EBP (CCAAT/enhancer-binding protein)-binding site in the promoters is essential for the IkappaB-zeta-mediated transcriptional activation. Using an artificial promoter consisting of the NF-kappaB- and C/EBP-binding sites, transcriptional activation was observed upon co-transfection with IkappaB-zeta and NF-kappaB, indicating that these sequences are minimal elements that confer the IkappaB-zeta-mediated transcriptional activation. Chromatin immunoprecipitation assays and knockdown experiments showed that both IkappaB-zeta and the NF-kappaB subunits were recruited to the NGAL promoter and were essential for the transcriptional activation of the hBD-2 and NGAL promoters on stimulation with IL-1beta. The activation of the NGAL promoter by transfection of IkappaB-zeta and NF-kappaB was suppressed in C/EBPbeta-depleted cells. Thus IkappaB-zeta acts as an essential transcriptional activator by forming a complex with NF-kappaB on promoters harbouring the NF-kappaB- and C/EBP-binding sites, upon stimulation of TLRs or IL-1 receptor.

The nuclear IκB protein IκB-ζ is barely detectable in resting cells and is induced in macrophages and fibroblasts following stimulation of innate immunity via Toll-like receptors. The induced IκB-ζ associates with nuclear factor (NF)-κB in the nucleus and plays crucial roles in its transcriptional regulation. Here, we examined the induction of IκB-ζ in B lymphocytes, one of the major players in adaptive immunity. Upon crosslinking of the surface immunoglobulin complex, IκB-ζ mRNA was robustly induced in murine B-lymphoma cell line A20 cells. While the crosslinking activated NF-κB and induced its target gene, IκB-α, co-crosslinking of Fcγ receptor IIB to the surface immunoglobulin complex inhibited NF-κB activation and the induction of IκB-ζ and IκB-α, suggesting critical roles for NF-κB in the induction. These results indicate that IκB-ζ is also induced by stimulation of B cell antigen receptor, suggesting that IκB-ζ is involved in the regulation of adaptive immune responses.

Posttranscriptional regulatory elements in the 3′-untranslated region (UTR) of mRNAs play an important role in mRNA stabilization. Induction of IκB-ζ, a critical transcriptional regulator in the innate immune response, is mediated via specific mRNA stabilization by lipopolysaccharide (LPS) and interleukin (IL)-1β. It is known that the 3′-UTR of IκB-ζ, especially 165 nucleotides after the stop codon, plays a crucial role in mRNA stability. Herein, we show that AU-rich elements and miRNA targets in these 165 3′-UTR nucleotides are dispensable for stability of IκB-ζ mRNA. Additionally, NF-κB activation is important for IκB-ζ transcription, but dispensable for IκB-ζ mRNA stability. Interestingly, high-throughput screening results show that MyD88, a signal molecule responsive to LPS/IL-1β stimulation, is key for stabilizing IκB-ζ mRNA expression. Moreover, MyD88-deficient macrophages exhibited a decreased half-life of IκB-ζ mRNA expression. These results indicate that the LPS/IL-1β-MyD88 axis plays a crucial role for stabilization of IκB-ζ mRNA.

Innate immunity responds to various pathogen-associated molecular patterns (PAMPs) to evaluate the biological nature of foreign materials by using limited numbers of receptors. Analyses of interactions between PAMPs and its receptors are essential to understand the molecular basis regarding how we discriminate self and non-self materials. Upon infection of horseshoe crabs, an arthropod species, rapid hemolymph coagulation is induced to engulf invading microorganisms by a cascade-type reaction. The reaction is very sensitive to lipopolysaccharide and (1-->3)-beta-D-glucans on Gram-negative bacteria and fungi, respectively, and hence is utilized as assay reagents that detect and quantitate these PAMPs with a name of "limulus test." In this mini-review, recognition of (1-->3)-beta-D-glucans by a unique serine protease zymogen factor G of horseshoe crab is described. Molecular dissection and detailed kinetic analyses have revealed that multivalent binding to polymers of a simple target structure is one of the principles that allows stable and specific recognition of PAMPs by pattern recognition receptors in innate immunity.

We have recently identified an inducible nuclear factor-kappaB (NF-kappaB) regulator, IkappaB-zeta, which is induced by microbial ligands for Toll-like receptors such as lipopolysaccharide and the proinflammatory cytokine interleukin (IL)-1beta but not by tumor necrosis factor (TNF)-alpha. In the present study, we examined mechanisms for stimulus-specific induction of IkappaB-zeta. The analysis of the IkappaB-zeta promoter revealed an essential role for an NF-kappaB binding sequence in transcriptional activation. The activation, however, did not account for the Toll-like receptor/IL-1 receptor-specific induction of IkappaB-zeta, because the promoter analysis and nuclear run-on analysis indicated that its transcription was similarly induced by TNF-alpha. To examine post-transcriptional regulation, we analyzed the decay of IkappaB-zeta mRNA, and we found that it was specifically stabilized by lipopolysaccharide or IL-1beta but not by TNF-alpha. Furthermore, we found that costimulation with TNF-alpha and another proinflammatory cytokine, IL-17, elicited the IkappaB-zeta induction. Stimulation with IL-17 alone did not induce IkappaB-zeta but stabilized its mRNA. Therefore, IkappaB-zeta induction requires both NF-kappaB activation and stimulus-specific stabilization of its mRNA. Because IkappaB-zeta is essential for expression of a subset of NF-kappaB target genes, the stimulus-specific induction of IkappaB-zeta may be of great significance in regulation of inflammatory reactions.

Toll-like receptor 4 (TLR4) and MD-2 are pivotal components that elicit inflammatory responses to lipopolysaccharide (LPS). They have been shown to form a physical complex on the cell surface that responds directly to LPS. However, the functional region of TLR4 required for association with MD-2 and LPS responsiveness is poorly understood. To identify the region of TLR4, we created a series of mutants with deletions in the extracellular domain and examined their activities in human embryonic kidney 293 cells. A mutant with a 317-amino acid deletion from the membrane proximal region of TLR4 was capable of associating with MD-2, while only a 9-amino acid truncation of the N terminus severely impaired the interaction. The association between the two molecules was well correlated with TLR4 maturation into an endoglycosidase H-resistant form and the cell surface expression. Mouse MD-2 bound to human TLR4, but its activity to facilitate the cell surface expression of TLR4 and confer LPS responsiveness was much weaker than that of human MD-2, indicating species specificity. A chimeric receptor composed of the N-terminal region of human TLR4 and the adjacent region of mouse TLR4 showed preference for human MD-2 in its transport to the cell surface and responsiveness to LPS. Taken together, the N-terminal region of TLR4 is essential for association with MD-2, which is required for the cell surface expression and hence the responsiveness to LPS.

Gastric epithelial cells were incubated with a panel of clinical isolates of Helicobacter pylori, including nonulcer dyspepsia with gastritis (HS, n = 20), gastric ulcer (HU, n = 20), duodenal ulcer (HD, n = 21), and gastric cancer (HC, n = 20). HC strains induced a higher cyclooxygenase-2 (COX-2) expression than those from HS, HD, and HU. The bacterial virulence factors and the host cellular pathways were investigated. Virulence genes of iceA, vacA, babA2, cagA 3' repeat region, and hrgA failed to show any association with the disease status and COX-2 expression. Methylation-specific polymerase chain reaction revealed HC strains not affecting the methylation status of COX-2 promoter. Nuclear factor (NF)-kappaB, NF-interleukin 6, and cAMP response element were found to be involved in COX-2 induction. We explored a novel NF-kappaB activation pathway. The mutants of TLR2 and TLR9, but not TLR4, inhibited H. pylori-induced COX-2 promoter activity, and neutralizing antibodies for TLR2 and TLR9 abolished H. pylori-induced COX-2 expression. Phosphatidylinositol-specific phospholipase C (PI-PLC), protein kinase C (PKC), and Src inhibitors inhibited COX-2 induction. The dominant-negative mutants of NIK and various IkappaB kinase complexes, including IKKbeta (Y188F), IKKbeta (Y199F), and IKKbeta (FF), inhibited the COX-2 promoter activity. Phosphorylation of GST-IKKbeta (132-206) at Tyr188 and Tyr199 by c-Src was found after H. pylori infection. In summary, H. pylori induces COX-2 expression via activations of NF-kappaB, NF-interleukin 6, the cAMP response element. In NF-kappaB activation, H. pylori acts through TLR2/TLR9 to activate both the cascade of PI-PLCgamma/PKCalpha/c-Src/IKKalpha/beta and the cascade of NIK/IKKalpha/beta, resulting in the IkappaBalpha degradation and the expression of COX-2 gene. The COX-2 overexpression may contribute to the carcinogenesis in patients colonized with these strains.

Activation of nuclear factor-kappaB (NF-kappaB), a prominent cellular response to bacterial endotoxin or other microbial products, must be strictly regulated because excessive activation leads to overproduction of cytotoxic cytokines that culminates in septic shock. During screening for genes up-regulated upon inflammation, we identified a new member of the IkappaB family proteins with the ankyrin-repeats. This protein, designated IkappaB-zeta, is hardly detectable in resting cells, but is strongly induced upon stimulation by lipopolysaccharide, which stimulates cells through the Toll-like receptor 4. Interleukin-1beta stimulation also results in the strong induction of IkappaB-zeta, but tumor necrosis factor-alpha does not. In contrast to IkappaB-alpha or IkappaB-beta, IkappaB-zeta localizes in the nucleus, where it inhibits NF-kappaB activity. NF-kappaB activity is essential for the induction of IkappaB-zeta, but is not sufficient. Thus, this protein is a new anti-inflammatory protein, which is specifically induced upon inflammation to regulate NF-kappaB activity.

CD14 is a protein that mediates lipopolysaccharide (LPS)-induced biological responses such as activation of a transcriptional factor, nuclear factor (NF)-κB. It exists as a soluble form (sCD14) in serum and mediates LPS responses of epithelial and endothelial cells as well as a membrane-bound form (mCD14) on monocytes and macrophages. To obtain sCD14 in large quantity for its structural and functional characterization, we expressed the full-length form of human recombinant sCD14 (rsCD14) in a methylotrophic yeast, Pichia pastoris. The recombinant protein was expressed as a major protein in the culture supernatant and purified by ammonium sulfate precipitation, followed by three steps of ion exchange chromatographies. Finally, 1.6 mg of the protein was obtained in high purity from 2 L of the supernatant and its identity to sCD14 was confirmed by NH2-terminal amino acid sequence analysis. The purified protein was found to have N-linked sugars by an analysis of enzymatic deglycosylation. A native PAGE analysis revealed that the protein was able to form complexes with LPS. In addition, the rsCD14 protein could mediate the LPS-mediated activation of NF-κB in human embryonic kidney 293 cells transfected with Toll-like receptor 4 and MD-2, indicating that the purified protein is biologically active. Thus, the rsCD14 protein expressed in P. pastoris and highly purified in a large amount is useful for its structural and functional studies.

IκB-ζ, a new negative-regulator of nuclear factor-κB (NF-κB), is strongly induced by lipopolysaccharide or interleukin-1β stimulation, but not by tumor necrosis factor-α. Here, we analyzed the mechanisms for transcriptional induction of IκB-ζ. IκB-ζ mRNA was induced by overexpression of MyD88 or TRAF6, but not TRAF2. Stimulation of macrophages with peptidoglycan or CpG DNA, which activated Toll-like receptor 2 or 9, respectively, also resulted in IκB-ζ induction. Thus, activation of the MyD88-dependent signaling pathway, commonly found downstream of different Toll/interleukin-1 receptor (TIR) domains, is sufficient for IκB-ζ induction. The induction was inhibited by treatment with various inhibitors of NF-κB activation or by overexpressing IκB-α or β, indicating essential roles for NF-κB in IκB-ζ induction. However, overexpression of the NF-κB subunits induced IκB-α, but not IκB-ζ. These results indicate the existence of another signal essential for IκB-ζ induction, which is specifically mediated by the TIR domain-mediated signaling pathway.

Although (1-->3)-beta-d-glucans, which are one of major fungal cell wall components, are known to activate invertebrate innate immune systems, their activities on mammalian cells remain elusive. Here, we report their activities on mouse macrophages. Among the various (1-->3)-beta-d-glucans, curdlan, a linear (1-->3)-beta-d-glucan, although not branched beta-glucans, exhibits significant activity to stimulate nuclear factor-kappaB in macrophages. The activity of curdlan is dramatically enhanced by pretreatment with sodium hydroxide or dimethyl sulfoxide, which disrupts multiple-stranded helices of (1-->3)-beta-d-glucans, and is dose-dependently inhibited by a (1-->3)-beta-d-glucan-binding protein and by laminarioligosaccharides with (1-->3)-beta-d-glucosidic linkages. Intriguingly, the activity of curdlan is also augmented by incubation with zymolyase, which releases (1-->3)-beta-d-glucans with a single helical structure from the glucan-networks assembled by multiple-stranded helices. The activation of macrophages culminates in the production of inducible nitric-oxide synthase, tumor necrosis factor-alpha, and macrophage inflammatory protein-2. Furthermore, a dominant-negative mutant of MyD88, an adaptor protein mediating signaling through the Toll-like receptor/inerleukin-1 receptor-like (TIR) domain, inhibits the activation of macrophages by curdlan. These results strongly suggest that macrophages respond to linear (1-->3)-beta-d-glucans, possibly released from fungal cell walls, via a receptor(s) harboring the TIR domain, such as a Toll-like receptor, to induce inflammatory reactions.

The horseshoe crab factor G, a heterodimeric serine protease zymogen, is activated by (1-->3)-beta-D-glucan on fungal cell walls. The activation initiates the hemolymph-clotting cascade, a critical reaction for the defense against microorganisms. In the present study, we identified the domain responsible for the glucan recognition by factor G and characterized its interaction with (1-->3)-beta-d-glucan and its derivatives. Among three domains in subunit alpha of factor G, identified as the glucan-binding domain, was the COOH-terminal xylanase Z-like domain composed of two tandem-repeating units, each of which exhibits sequence similarities to the cellulose-binding domains of bacterial xylanases. Each of the single units bound to the glucan with lower affinities, and the association constant increased two orders with the tandem-repeating structure (K(a) = 8.0 x 10(8) m(-1)). In addition to longer glucans, (1-->3)-beta-D-glucan oligosaccharides incapable of activating factor G bound also to factor G and competitively inhibited the zymogen activation. The minimum structure required for the binding was a (1-->3)-beta-d-glucan disaccharide, indicating that conformation-dependent structures are not essential for the recognition. Therefore, increasing avidity by multivalent binding sites with low affinities to simple structures on biologically active polymers may be one of the principles that allows stable and specific recognition of pathogens by pattern recognition receptors in innate immunity.

Bisphenol A diglycidyl ether (BADGE) is a newly described peroxisome proliferator-activated receptor gamma (PPARgamma) antagonist in adipogenic cells. In contrast, in the macrophage-like cell line RAW 264.7, BADGE, like the PPARgamma agonist pioglitazone hydrochloride, not only increased promoter activity of the PPARgamma-luciferase reporter gene, but also suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) production. These results suggest that BADGE is a PPARgamma agonist in RAW 264.7 cells. Furthermore, overexpression of the coactivator p300 restored BADGE- or pioglitazone hydrochloride-suppressed promoter activity of the nuclear factor-kappa B (NF-kappaB)-luciferase reporter gene, suggesting that PPARgamma may interfere with NF-kappaB transcriptional activity via coactivator competition.

The transcription factor nuclear factor-κB (NF-κB) plays crucial roles in a wide variety of cellular functions and its activity is strictly regulated by cytosolic inhibitors known as IκBs. We here report a new member of the IκB protein family, IκB-ζ, harboring six ankyrin repeats at its carboxyl terminus. IκB-ζ mRNA is strongly induced after stimulation by lipopolysaccharide. The induction of IκB-ζ is also observed by stimulation with interleukin-1β but not by tumor necrosis factor-α. In contrast to cytosolic IκB-α, -β, and -e, the induced IκB-ζ localizes in the nucleus via its amino-terminal region, which shows no homology with other proteins. Transiently expressed IκB-ζ inhibits the NF-κB activity without affecting the nuclear translocation of NF-κB upon stimulation. The expressed IκB-ζ preferentially associates with the NF-κB subunit p50 rather than p65 and recombinant IκB-ζ proteins inhibit the DNA binding of the p65/p50 heterodimer and the p50/p50 homodimer. Thus, IκB-ζ negatively regulates NF-κB activity in the nucleus, possibly in order to prevent excessive inflammation. Moreover, transfection of IκB-ζ renders cells more susceptible to apoptosis induced by tumor necrosis factor-α. The proapoptotic activity of IκB-ζ further suggests that it might be one of key regulators for inflammation and other biologically relevant processes.

Activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), which forms a heterodimer with retinoic X receptor (RXR), inhibit the production of certain inflammatory mediators. To clarify the role of the PPAR gamma:RXR signaling pathway in Kupffer cells, we studied the effect of an RXR agonist and PPARgamma agonist on LPS-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production. An RXR-specific agonist, Ro47-5944, and a PPAR gamma-specific agonist, AD4833 (pioglitazone hydrochloride), each inhibited LPS-induced NO and TNF-alpha production. The combined treatment of Ro47-5944 and AD4833 resulted in enhanced inhibition, and suppressed the mRNA levels of NO and TNF-alpha. PPAR gamma:RXR activation did not affect the level of LPS-induced phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase. PPAR gamma:RXR activation also did not affect nuclear factor kappa B (NF-kappa B) nuclear translocation nor NF-kappa B and activator protein 1 (AP-1) activation in the electrophoretic mobility-shift assay. Finally, PPAR gamma:RXR activation suppressed the LPS-induced promoter activity of the NF-kappa B-luciferase reporter gene in RAW 264.7 cells. These data imply that PPARgamma:RXR activation suppresses LPS-induced NO and TNF-alpha production in Kupffer cells, and that this inhibition occurred at the transcriptional level. Although no consensus PPAR gamma:RXR-responsive element in the promoter regions of the inducible isoform of nitric oxide synthase (iNOS) and TNF-alpha genes was found, PPAR gamma:RXR may interfere with NF-kappa B and AP-1 transcriptional activity. Our data also suggest a potential therapeutic approach for moderating hepatic injury such as endotoxin shock in which Kupffer cell activation has been implicated.

A clottable protein coagulogen of the horseshoe crab Tachypleus tridentatus is proteolytically converted into an insoluble coagulin gel through non-covalent self-polymerization. Here we identified binding sites for the polymerization. A tryptic fragment, derived from the coagulin polymer chemically cross-linked by a bifunctional cross-linker, was isolated. Amino acid sequence analysis indicated that the fragment consists of two peptides cross-linked between Lys(85) and Lys(156). The two lysine residues are oppositely located at the head and tail regions of the elongated molecule separated by a much greater distance than the length of the cross-linker, which suggests that the cross-linking occurs intermolecularly. Based on the x-ray structural analysis, exposure of a hydrophobic cove on the head in response to the release of peptide C has been postulated (Bergner, A., Oganessyan, V., Muta, T., Iwanaga, S., Typke, D., Huber, R., and Bode, W. (1996) EMBO J. 15, 6789-6797). An octapeptide containing Tyr(136), which occupies the tail end of coagulin, was found to inhibit the polymerization. Replacement of Tyr(136) of the peptide with Ala resulted in loss of the inhibitory activity. These results indicated that the polymerization of coagulin proceeds through the interaction between the newly exposed hydrophobic cove on the head and the wedge-shaped hydrophobic tail.

We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus , which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a horseshoe crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of “bouquet arrangement” of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The horseshoe crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the horseshoe crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.

Invertebrate animals, which lack adaptive immune systems, have developed defense systems, so-called innate immunity, that respond to common antigens on the surface of potential pathogens. One such defense system is involved in the cellular responses of horseshoe crab hemocytes to invaders. Hemocytes contain two types, large (L) and small (S), of secretory granules, and the contents of these granules are released in response to invading microbes via exocytosis. Recent biochemical and immunological studies on the granular components of L- and S-granules demonstrated that the two types of granules selectively store granule-specific proteins participating in the host defense systems. L-Granules contain all the clotting factors essential for hemolymph coagulation, protease inhibitors including serpins and cystatin, and anti-lipopolysaccharide (LPS) factor and several tachylectins with LPS binding and bacterial agglutinating activities. On the other hand, S-granules contain various new cysteine-rich basic proteins with antimicrobial or bacterial agglutinating activities, such as tachyplesins, big defensin, tachycitin, and tachystatins. The co-localization of these proteins in the granules and their release into the hemolymph suggest that they serve synergistically to construct an effective host defense system against invaders. Here, the structures and functions of these new types of defense molecules found in the Japanese horseshoe crab (Tachypleus tridentatus) are reviewed.

A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a β-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned. The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolylcis/trans-isomerase. p27 exhibits isomerase activity with ak cat /K m of 0.18 μm −1 s−1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506. Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion. Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation. These results indicate that p27 is a new peptidyl-prolylcis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules.

Whereas accumulating evidence indicates that a number of inflammatory genes are induced by activation of nuclear factor-κB and other transcription factors, less is known about genes that are suppressed by proinflammatory stimuli. Here we show that expression of thioredoxin-interacting protein (Txnip) is dramatically suppressed both in mRNA and protein levels upon stimulation with lipopolysaccharide in mouse and human macrophages. In addition to lipopolysaccharide, a Toll-like receptor 4 ligand, stimulation with other Toll-like receptor ligands such as CpG DNA also suppressed Txnip expression. Not only the Toll-like receptor ligands, but also other proinflammatory stimulators, such as interleukin-1β and tumor necrosis factor-α elicited the similar response in fibroblasts. Suppression of Txnip by lipopolysaccharide is accompanied by a decrease of the glucose sensing transcription factor MondoA in the nuclei and dissociation of the MondoA:Mlx complex that bound to the carbohydrate-response elements in the Txnip promoter in unstimulated cells. Lipopolysaccharide-mediated decrease of nuclear MondoA was inhibited in the presence of 2-deoxyglucose. Furthermore, blockage of glyceraldehyde-3-phosphate dehydrogenase by iodoacetate alleviated the suppression of Txnip mRNA by lipopolysaccharide, suggesting the involvement of glucose-metabolites in the regulation. Since Txnip is implicated in the regulation of glucose metabolism, this observation links between inflammatory responses and metabolic regulation.

Invertebrate animals, which lack adaptive immune systems, have developed defense systems that respond to common antigens on the surface of potential pathogens. Hemolymph coagulation is one such defense system in innate immunity. The discovery of lipopolysaccharide-sensitive and (1→3)β- d -glucan-sensitive serine protease zymogens in horseshoe crab (limulus) hemocytes, both of which trigger the coagulation cascade, has exemplified how the animals detect and respond to foreign materials.

Horseshoe crab hemocyte lysate responds to (1 → 3)-β-D-glucans, initiating an enzymatic cascade, which culminates in clot formation.We have purified to homogeneity the serine protease zymogen factor G, which is directly activated by (1 → 3)-β-D-glucans and which initiates the hemolymph clotting cascade. Factor G is a heterodimeric protein composed of two noncovalently associated subunits α (72 kDa) and β (37 kDa). In the presence of (1 → 3)-β-D-glucans such as curdlan and paramylon, factor G is autocatalytically activated to an active serine protease named factor G. This activation is accompanied by limited proteolysis of both subunits: the 72-kDa subunit α is cleaved to 55-kDa and 17-kDa fragments, and the 37-kDa subunit β is shortened to 34 kDa. Longer incubations with (1 → 3)-β-D-glucans result in cleavage of the 55-kDa fragment to 46 kDa and the 34-kDa fragment to 32 kDa, with concomitant loss of amidase activity. Reconstitution experiments using purified proteins participating in the hemolymph clotting cascade demonstrate that factor G is capable of activating proclotting enzyme directly, resulting in the conversion of coagulogen to coagulin gel. Thus, purified factor G is shown to be the primary initiator of the (1 → 3)-β-D-glucan-sensitive coagulation pathway in the horseshoe crab hemocyte lysate.

The Fc receptor on B lymphocytes, Fc gamma RIIB (beta 1 isoform), helps to modulate B-cell activation triggered by the surface immunoglobulin complex. Crosslinking of membrane immunoglobulin by antigen or anti-Ig F(ab')2 antibody induces a transient increase in cytosolic free Ca2+, a rise in inositol-3-phosphate, activation of protein kinase C, and enhanced protein tyrosine phosphorylation. Crosslinking Fc gamma RIIB with the surface immunoglobulin complex confers a dominant signal that prevents or aborts lymphocyte activation triggered through the ARH-1 motifs of the signal transduction subunits Ig-alpha and Ig-beta. Here we show that Fc gamma RIIB modulates membrane immunoglobulin-induced Ca2+ mobilization by inhibiting Ca2+ influx, without changing the pattern of tyrosine phosphorylation. A 13-amino-acid motif in the cytoplasmic domain of Fc gamma RIIB is both necessary and sufficient for this effect. Tyrosine at residue 309 in this motif is phosphorylated upon co-crosslinking with surface immunoglobulin; mutation of this residue aborts the inhibitory effect of Fc gamma RIIB. This inhibition is directly coupled to signalling mediated through Ig-alpha and Ig-beta as evidenced by chimaeric IgM/alpha and IgM/beta molecules. The 13-residue motif in Fc gamma RIIB controls lymphocyte activation by inhibiting a Ca2+ signalling pathway triggered through ARH-1 motifs as a result of recruitment of novel SH2-containing proteins that interact with this Fc gamma RIIB cytoplasmic motif.

IκB-ζ, an essential inflammatory regulator, is specifically induced by Toll-like receptor ligands or interleukin (IL)-1β by post-transcriptional activation mediated via a 165-nucleotide element in IκB-ζ mRNA. Here, we analyzed the Toll-like receptor-IL-1 receptor signaling components involved in the post-transcriptional regulation of IκB-ζ with mutated estrogen receptor [ER(T2)] fusion proteins. Upon 4-hydroxytamoxifen treatment, the ER(T2) fusion proteins with IL-1 receptor-associated kinase (IRAK)1 and IRAK4 elicited specific activation of a reporter gene for the post-transcriptional regulation of IκB-ζ. The tumor necrosis factor receptor-associated factor (TRAF)6-ER(T2) protein activated nuclear factor-κB, but not post-transcriptional regulation, indicating that activation of IRAK1/4, but not of TRAF6, is sufficient to activate the 165-nucleotide element-mediated post-transcriptional mechanism. Interestingly, the post-transcriptional mechanism was not activated in TRAF6-deficient cells, indicating an essential role for TRAF6. Thus, the signaling pathway leading to nuclear factor-κB activation and the post-transcriptional activation bifurcates at IRAK1, suggesting a new pathway activated by IRAK1.

Plasmodium falciparum glycosylphosphatidylinositols (GPIs) have been proposed as malaria pathogenic factors based on their ability to induce proinflammatory responses in macrophages and malaria-like symptoms in mice. Parasite GPIs induce the production of inflammatory cytokines by activating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways. Importantly, inhibition of the extracellular-signal-regulated kinase (ERK) pathway upregulates a subset of cytokines, including IL-12. We investigated the role of nuclear transcription factor, IκB-ζ, in the GPI-induced dysregulated expression of IL-12 on inhibition of the ERK pathway. GPIs efficiently induced the expression of IκB-ζ in macrophages regardless of whether cells were pretreated or untreated with ERK inhibitors, indicating that ERK has no role in IκB-ζ expression. However, on ERK inhibition followed by stimulation with GPIs, NF-κB binding to Il12b promoter κB site was markedly increased, suggesting that the ERK pathway regulates Il12b transcription. Knockdown of IκB-ζ using siRNA markedly reduced the GPI-induced IL-12 production and abrogated the dysregulated IL-12 production in ERK inhibited cells. Together these results demonstrate that ERK modulates IL-12 expression by regulating IκB-ζ-dependent binding of NF-κB transcription factors to Il12b gene promoter. Additionally, our finding that IκB-ζ can be knocked down efficiently in primary macrophages is valuable for studies aimed at gaining further insights into IκB-ζ function. © 2011 IUBMB, IUBMB Life,, 2012

We designed a method for separating two types of granules, a smaller (S) but dense and a larger (L) but less dense granule from hemocytes of the horseshoe crab (Tachypleus tridentatus), using continuous sucrose density gradient centrifugation. The isolated L-granules contained at least three clotting factors plus a clottable protein, coagulogen, as the major component. The known anti-lipopolysaccharide factor and 7 additional unknown protein components were also present in the L-granules. Two known natural substrates, Pro-rich protein and 8.6 kDa protein, for limulus transglutaminase [Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., & Davie, E.W. (1993) J. Biol. Chem. 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachyplesins I and II (17 amino acids in length) as the major component, in addition to 6 unidentified proteins with molecular masses of less than 30 kDa. The structural analyses of tachyplesin analogs indicated that all these peptides of mature form are stored in the S-granules, together with a processing intermediate containing the COOH-terminal Gly-Lys sequence. We also found an Arg-rich protein of 22 kDa and a Leu-rich protein of 30 kDa in S-granules. Based on these observations, we speculate that protein components in L-granules, which probably contain all the factors essential for the limulus clotting system, participate in immobilization of invading microbes and that factors in the S-granules containing tachyplesins contribute to a self-defense system against invaders.

IκBζ, encoded by Nfibiz , is a nuclear IκB-like protein harboring ankyrin repeats. IκBζ has been shown to regulate IL-6 production in macrophages and Th17 development in T cells. However, the role of IκBζ in natural killer (NK) cells has not be understood. In the present study, we found that the expression of IκBζ was rapidly induced in response to IL-18 in NK cells, but not in T cells. Analysis of Nfkbiz −/− mice revealed that IκBζ was essential for the production of IFN-γ production and cytotoxic activity in NK cells in response to IL-12 and/or IL-18 stimulation. IL-12/IL-18–mediated gene induction was profoundly impaired in Nfkbiz −/− NK cells. Whereas the phosphorylation of STAT4 was normally induced by IL-12 stimulation, STAT4 was not recruited to the Ifng gene regions in Nfkbiz −/− NK cells. Acetylation of histone 3 K9 on Ifng regions was also abrogated in Nfkbiz −/− NK cells. IκBζ was recruited on the proximal promoter region of the Ifng gene, and overexpression of IκBζ together with IL-12 activated the Ifng promoter. Furthermore, Nfkbiz −/− mice were highly susceptible to mouse MCMV infection. Taken together, these results demonstrate that IκBζ is essential for the activation of NK cells and antiviral host defense responses.

Limulus (horseshoe crab) hemolymph is known to be very sensitive to bacterial endotoxin (LPS), which causes a rapid coagulation response. Hemolymph contains a single type of hemocyte that undergoes aggregation, adhesion, and degranulation in response to LPS. The granule contents are released into the hemolymph, where they form an insoluble gel. We have characterized four components involved in this coagulation response that comprise a cascade of three serine protease zymogens (factor C, factor B, and proclotting enzyme) and one clottable protein (coagulogen). Of these components, factor C sensitive to LPS is a protein composed of five complement-related domains ("Sushi" or SCR), an EGF-like domain, and a C-type lectinlike domain as well as a putative amino-terminal LPS-binding domain. This domain structure is very similar to that of selectin family of cell adhesion molecules, suggesting that it might also function as a cell adhesion molecule after the release into the hemolymph. Factor B and the proclotting enzyme share a common Cys-rich motif ("cliplike" domain) in the amino-terminal portions. This domain is also found in a putative serine protease zymogen ("easter") in Drosophila, which is essential for normal embryonic development. All four of the components of the cascade and an antibacterial protein (anti-LPS factor) are localized to a specific type of the hemocyte granule. Another antibacterial peptide (tachyplesins I and II) is localized in a distinct granule population. The contents of both granule populations are released into the hemolymph in response to LPS, where they cooperate in immobilization and killing of Gram-negative bacteria.

Innate immunity against microbial pathogens relies on the pattern recognition of cell wall components on invading microbes. Recent evidence has shown that a mammalian Toll-like receptor (TLR) is activated by bacterial lipopolysaccharides (LPS). The innate immunity in invertebrates is also triggered by LPS, as seen in the hemolymph coagulation in horseshoe crab. We report the cloning of a TLR from the Japanese horseshoe crab Tachypleus tridentatus. A cDNA coding for Tachypleus Toll was isolated from a hemocyte cDNA library and the open reading frame codes for a proprotein including a signal sequence. Like Drosophila Toll, Tachypleus Toll is a type I transmembrane protein with an extracellular domain consisting of two leucine-rich repeats flanked by two cystein-rich clusters and a cytoplasmic domain exhibiting striking similarity with the cytoplasmic domain of interleukin-1 receptor. Tachypleus Toll is most similar to Drosophila Toll in the domain architecture and the overall length.

Interleukin (IL)-17-producing helper T (T(H)17) cells are a distinct T-cell subset characterized by its pathological role in autoimmune diseases. IL-6 and transforming growth factor-beta (TGF-beta) induce T(H)17 development, in which the orphan nuclear receptors, RORgammat and RORalpha, have an indispensable role. However, in the absence of IL-6 and TGF-beta, the ectopic expression of RORgammat or RORalpha leads to only a modest IL-17 production. Here we identify a nuclear IkappaB family member, IkappaBzeta (encoded by the Nfkbiz gene), as a transcription factor required for T(H)17 development in mice. The ectopic expression of IkappaBzeta in naive CD4(+) T cells together with RORgammat or RORalpha potently induces T(H)17 development, even in the absence of IL-6 and TGF-beta. Notably, Nfkbiz(-/-) mice have a defect in T(H)17 development and a resistance to experimental autoimmune encephalomyelitis (EAE). The T-cell-intrinsic function of IkappaBzeta was clearly demonstrated by the resistance to EAE of the Rag2(-/-) mice into which Nfkbiz(-/-) CD4(+) T cells were transferred. In cooperation with RORgammat and RORalpha, IkappaBzeta enhances Il17a expression by binding directly to the regulatory region of the Il17a gene. This study provides evidence for the transcriptional mechanisms underlying T(H)17 development and points to a molecular basis for a novel therapeutic strategy against autoimmune disease.

Proinflammatory responses induced by Plasmodium falciparum glycosylphosphatidylinositols (GPIs) are thought to be involved in malaria pathogenesis. In this study, we investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs. We show that MK2 differentially regulates the GPI-induced production of TNF-alpha and IL-12. Although TNF-alpha production was markedly decreased, IL-12 expression was increased by 2-3-fold in GPI-stimulated MK2(-/-) macrophages compared with wild type (WT) cells. MK2(-/-) macrophages produced markedly decreased levels of TNF-alpha than WT macrophages mainly because of lower mRNA stability and translation. In the case of IL-12, mRNA was substantially higher in MK2(-/-) macrophages than WT. This enhanced production is due to increased NF-kappaB binding to the gene promoter, a markedly lower level expression of the transcriptional repressor factor c-Maf, and a decreased binding of GAP-12 to the gene promoter in MK2(-/-) macrophages. Thus, our data demonstrate for the first time the role of MK2 in the transcriptional regulation of IL-12. Using the protein kinase inhibitors SB203580 and U0126, we also show that the ERK and p38 pathways regulate TNF-alpha and IL-12 production, and that both inhibitors can reduce phosphorylation of MK2 in response to GPIs and other toll-like receptor ligands. These results may have important implications for developing therapeutics for malaria and other infectious diseases.

The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 μm in diameter) and less electron-dense than the D-granules (less than 0.6 μm in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.

Tachyplesins and their analogs are antimicrobial peptides composed of 17 or 18 amino acid residues present abundantly in acid extracts of hemocyte debris of horseshoe crabs. We purified here tachyplesin isopeptides from hemocytes of two species of Southeast Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus gigas, and determined their amino acid sequences. The major tachyplesin isolated from both species was identified, respectively, as tachyplesin I, which had previously been found in hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus). The yield from both species was very high (more than 70 mg per 100 g wet weight of hemocytes), i.e., comparable with that from T. tridentatus. In addition to tachyplesin I, a new tachyplesin isopeptide, named tachyplesin III, was also isolated from T. gigas hemocytes, in which an arginine replaced the 15th lysine of tachyplesin I. The carboxyl-terminal residue of the isolated tachyplesins I and III was confirmed, respectively, to be an arginine alpha-amide by chemical analysis. Furthermore, a tachyplesin peptide derivative with a carboxyl-terminal extension of glycine-lysine was newly found in the hemocytes of C. rotundicauda. It appeared to be an intermediate derived from a tachyplesin precursor during processing to the mature form.

Haemocytes of the horseshoe crab (Limulus) contain a new family of arthropodous peptide antibiotics, termed the tachyplesin family. These cationic peptides are composed of 17-18 amino acid residues with a C-terminal arginine alpha-amide. Tachyplesin I takes on a fairly rigid conformation constrained by two disulphide bridges and adopts a conformation consisting of an antiparallel beta-sheet connected by a beta-turn. Isopeptides of tachyplesin I with amino acid replacements, tachyplesins II and III, and polyphemusins I and II have also been found in the haemocytes of the South-East Asian species and Limulus polyphemus. These peptides are present in abundance in the small granules of the haemocytes and inhibit strongly the growth of not only Gram-negative and Gram-positive bacteria but also fungi such as Candida albicans. Tachyplesin exists in the prepro form consisting of 77 residues; this precursor is probably processed by intracellular proteases and an amidation enzyme before incorporation into the small granules of the haemocytes. We examined the mode of action of tachyplesin I on biomembranes, comparing it with that of gramicidin S. Tachyplesin caused an efflux of K+ from Staphylococcus aureus and Escherichia coli cells similar to that caused by gramicidin S. Another antimicrobial substance, anti-LPS factor, has been isolated from haemocytes.