Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide

Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.     

Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents

Toll-like receptor (TLR) agonists are being developed for use as vaccine adjuvants and as stand-alone immunomodulators because of their ability to stimulate innate and adaptive immune responses. Among the most thoroughly studied TLR agonists are the lipid A molecules that target the TLR4 complex. One promising candidate, monophosphoryl lipid A, which is a derivative of lipid A from Salmonella minnesota, has proven to be safe and effective as a vaccine adjuvant in > 120,000 human doses. A new class of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates (AGPs), have been engineered specifically to target human TLR4 and are showing promise as vaccine adjuvants and as monotherapeutic agents capable of eliciting nonspecific protection against a wide range of infectious pathogens. In this review, the authors provide an update of the preclinical and clinical experiences with the TLR4 agonists, MPL (Corixa Corporation) adjuvant and the AGPs.     

Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents

Toll-like receptor (TLR) agonists are being developed for use as vaccine adjuvants and as stand-alone immunomodulators because of their ability to stimulate innate and adaptive immune responses. Among the most thoroughly studied TLR agonists are the lipid A molecules that target the TLR4 complex. One promising candidate, monophosphoryl lipid A, which is a derivative of lipid A from Salmonella minnesota, has proven to be safe and effective as a vaccine adjuvant in > 120,000 human doses. A new class of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates (AGPs), have been engineered specifically to target human TLR4 and are showing promise as vaccine adjuvants and as monotherapeutic agents capable of eliciting nonspecific protection against a wide range of infectious pathogens. In this review, the authors provide an update of the preclinical and clinical experiences with the TLR4 agonists, MPL^® (Corixa Corporation) adjuvant and the AGPs.     

Monocytes heterozygous for the Asp299Gly and Thr399Ile mutations in the Toll-like receptor 4 gene show no deficit in lipopolysaccharide signalling

Toll-like receptor 4 (TLR4)-mediated recognition of lipopolysaccharide (LPS) is required for efficient recognition of Gram-negative bacterial infections. Two commonly occurring mutations in the human TLR4 gene (Asp299Gly and Thr399Ile) have recently been shown to be associated with blunted physiological responses to inhaled LPS, and with increased risk of Gram-negative bacteraemia in sepsis patients and reduced risk of atherosclerosis in an Italian population. Here we show that monocytes from individuals heterozygous for both mutations in the TLR4 gene exhibit no deficit in recognition of LPS of Escherichia coli, Neisseria meningitidis, Bacteroides fragilis, Yersinia pestis, Chlamydia trachomatis, Porphyromonas gingivalis, or Pseudomonas aeruginosa. We propose that the relatively high frequency of these mutations in the Caucasian population may reflect modified responses of carriers to alternative TLR4 agonists.     

Investigating Toll-like receptor agonists for potential to treat hepatitis C virus infection

Toll-like receptors (TLRs) are key mediators of innate immunity, and their activation by microbial components leads to the production of cytokines and interferons. Recombinant alpha interferon has been used to treat several viral diseases and is the current standard of care for hepatitis C virus (HCV) infection. Recently, agonists of TLR7 and TLR9 have been shown to have clinical efficacy in HCV patients, and this is correlated with their ability to induce endogenous type I interferon production. We have carried out a comprehensive study of agonists of TLRs 1 to 9 to determine if any additional TLRs can induce antiviral molecules from human peripheral blood mononuclear cells (PBMCs). The agonists were incubated with PBMCs, and the supernatant was then removed and added to HCV replicon cells to assess antiviral activity. Agonists of TLRs 3, 4, 7, 8, and 9 were found to be potent inducers of antiviral activity in PBMC supernatants, and the activity correlated with the induction of alpha interferon and the interferon-induced antiviral biomarker 2',5'-oligoadenylate synthase. Antiviral activity of TLR7 and TLR8 agonists was blocked by an antibody that binds to the type I interferon receptor, confirming that the antiviral activity results from type I interferon induction. TLR4 and TLR8 agonists were found to strongly induce the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha at concentrations similar to those inducing antiviral activity. This raises concerns about adverse side effects if these were to be used as antiviral agents. We therefore conclude that TLRs 3, 7, and 9 represent the most attractive targets for the development of new HCV therapies.     

Inhibition of endotoxin response by e5564, a novel Toll-like receptor 4-directed endotoxin antagonist

Alpha-D-glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-beta-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt (E5564) is a second-generation synthetic lipodisaccharide designed to antagonize the toxic effects of endotoxin, a major immunostimulatory component of the outer cell membrane of Gram negative bacteria. In vitro, E5564 dose dependently (nanomolar concentrations) inhibited lipopolysaccharide (LPS)-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood as measured by production of tumor necrosis factor-alpha and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and has no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 microM. E5564 blocked LPS-mediated activation of nuclear factor-kappaB in toll-like receptor 4/MD-2-transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar to (6-O-[2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]-beta-O-phosphono-alpha-D-glucopyranose tetrasodium salt (E5531), another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose- and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for durations likely sufficient to permit clinical application. These results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.     

The toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells

The susceptibility to infections induced by Gram-negative bacteria is largely determined by innate immune responses to bacteria cell wall lipopolysaccharide (LPS). The stimulation of B cells by LPS enhances their antigen-presenting capacity and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing antibodies. Similar to macrophages and neutrophils, the LPS-induced activation of B cells is dependent on Toll-like receptor (TLR)4. Here, we demonstrate that the responses of B cells to LPS are also regulated by another TLR protein, RP105, which is predominantly expressed on mature B cells in mice and humans. The analysis of mice homozygous for the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS. Using originally LPS-unresponsive Ba/F3 cells expressing exogenous TLR4 and RP105, we demonstrate the functional cooperation between TLR4 and RP105 in LPS-induced nuclear factor kappaB activation. These data suggest the existence of the TLR4-RP105 signaling module in the LPS-induced B cell activation.     

Toll-like receptor 4, nitric oxide, and myocardial depression in endotoxemia

The molecular mechanisms that mediate gram-negative sepsis-associated myocardial dysfunction remain elusive. Myocardial expression of inflammatory mediators is Toll-like receptor 4 (TLR4) dependent. However, it remains to be elucidated whether TLR4, expressed on cardiac myocytes, mediates impairment of cardiac contractility after lipopolysaccharide (LPS) application. Cardiac myocyte contractility, measured as sarcomere shortening of isolated cardiac myocytes from C3H/HeJ (with nonfunctional TLR4) and C3H/HeN (control), were recorded at stimulation frequencies between 0.5 and 10 Hz and after incubation with 1 and 10 mug/mL LPS for up to 8 h. Control cells treated with LPS were investigated with and without a competitive LPS inhibitor (E5564) and a specific inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea. In control mice, LPS reduced sarcomere shortening amplitude and prolonged duration of relaxation, whereas sarcomere shortening of C3H/HeJ cells was insensitive to LPS. NFkappaB and iNOS were upregulated after LPS application in control mice compared with C3H/HeJ. Inhibition of TLR4 by E5564 as well as inhibition of iNOS prevented the influence of LPS on contractile activity in control myocytes. LPS-dependent suppression of cardiac myocyte contractility was significantly blunted in C3H/HeJ mice. Competitive inhibition of functional TLR4 with E5564 protects cardiac myocyte contractility against LPS. These findings suggest that TLR4, expressed on cardiac myocytes, contributes to sepsis-induced myocardial dysfunction. E5564, currently under investigation in two clinical phase II trials, seems to be a new therapeutic option for the treatment of myocardial dysfunction in sepsis associated with endotoxemia.     

Comparison of receptor mechanisms and efficacy requirements for delta-agonist-induced convulsive activity and antinociception in mice

Delta-opioid receptor-selective agonists produce antinociception and convulsions in several species, including mice. This article examines two hypotheses in mice: 1) that antinociception and convulsive activity are mediated through the same type of delta-receptor and 2) that greater delta-agonist efficacy is required for antinociception than for convulsive activity. Delta-mediated antinociception was evaluated in the acetic acid-induced abdominal constriction assay, which involves a low-intensity noxious stimulus; convulsive activity was indicated as a mild tonic-clonic convulsive episode followed by a period of catalepsy. In delta-opioid receptor knockout mice [DOR-1(-/-)], the nonpeptidic delta-agonists (+/-)-4-[(R*)-[(2S*,5R*)-2,5-dimethyl-4-(2-propenyl)-1- piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide hydrochloride (BW373U86) and (+)-4-[(R)-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)methyl]-N, N-diethylbenzamide (SNC80) failed to produce convulsive behavior demonstrating the absolute involvement of DOR-1 in this effect. In NIH Swiss mice expressing delta-opioid receptors, BW373U86 produced both antinociception and convulsive activity. These effects were antagonized by the putative delta(1)-receptor-selective antagonist 7-benzylidenenaltrexone and the putative delta(2)-receptor-selective antagonist naltriben. Tolerance developed to both the convulsive and antinociceptive effects of BW373U86. Tolerance to the convulsive, but not the antinociceptive, effects of BW373U86 was largely prevented when the antagonist naltrindole was given 20 min after each dose of the agonist in a 3-day treatment paradigm. The convulsive action of BW373U86 was also less sensitive than the antinociceptive action to treatment with the irreversible delta-antagonist naltrindole isothiocyanate. Collectively, these data suggest that the convulsive and antinociceptive activities of delta-agonists are mediated through the same receptor but that the receptor reserve for delta-mediated convulsive activity is greater than for delta-mediated antinociceptive activity.     

SYNERGISTIC INTERACTION OF MACROPHAGES AND LYMPHOCYTES IN ANTIGEN-INDUCED TRANSFORMATION OF LYMPHOCYTES

The role of macrophages and lymphocytes in antigen-induced transformation of lymphocytes has been investigated. Lymphocytes and macrophages were obtained from inbred strain 13 guinea pigs which were either unimmunized or immunized with complete Freund's adjuvant (CFA) or tetanus toxoid in CFA. The transformation response to PPD or tetanus toxoid was assayed by tritiated thymidine incorporation. Addition of macrophages to immune lymphocytes significantly increased their response to purified protein derivative (PPD) or tetanus toxoid. This was observed if the macrophages were (a) "immune" or "nonimmune", (b) unirradiated or irradiated (3000 R), (c) 99% pure, and (d) peritoneal or alveolar in origin. Neither immune nor nonimmune macrophages were able to induce nonimmune lymphocytes to respond to PPD or tetanus toxoid. When macrophages were incubated with PPD or tetanus toxoid and then washed, they stimulated immune lymphocytes to transform. An incubation time of ½ hr was adequate, however, 2–4: hr was optimal. These studies indicate (a) that antigen-induced transformation of lymphocytes is greatly enhanced by macrophages; (b) that macrophage-antigen interaction can antecede lymphocyte-antigen interaction and results in macrophages which are able to stimulate lymphocyte transformation; and (c) that the immunological memory requisite to elicit specific transformation responses is a property of the lymphocyte and not the macrophage.     

Stimulation of adenosine A2A receptor inhibits LPS-induced expression of intercellular adhesion molecule 1 and production of TNF-alpha in human peripheral blood mononuclear cells

LPS stimulates CD14/Toll-like receptor (TLR) 4, leading to induce TNF-alpha production. Cell-to-cell interaction through the engagement between intercellular adhesion molecule (ICAM) 1 on monocytes and its ligand on T cells has been suggested to play a role in the TNF-alpha production by LPS-treated human peripheral blood mononuclear cells (PBMCs). Adenosine is reported to inhibit LPS-induced TNF-alpha production. However, little is known about the mechanism of the inhibitory effects induced by adenosine on the LPS-induced immune responses. We found that adenosine inhibited the expression of ICAM-1 and the production of TNF-alpha by human PBMC via adenosine A2A receptor in the presence of LPS. However, the stimulation of A1R or A3R enhanced the actions of adenosine. Adenosine had no effect on the expression of CD14 and TLR-4, suggesting that the inhibitory effects of adenosine on the LPS actions might be independent of the expression of CD14 and TLR-4. Thus, adenosine differentially regulates the expression of ICAM-1 and the production of TNF-alpha through plural subtypes of receptors.     

CPG-7909 (PF-3512676, ProMune): toll-like receptor-9 agonist in cancer therapy

Stimulation of toll-like receptor (TLR)9 activates human plasmacytoid dendritic cells and B cells, and induces potent innate immune responses in preclinical tumor models and in patients. CpG oligodeoxynucleotides (ODNs) are TLR9 agonists that show promising results as vaccine adjuvants and in the treatment of cancers, infections, asthma and allergy. PF-3512676 (ProMune) was developed as a TLR9 agonist for the treatment of cancer as monotherapy and as an adjuvant in combination with chemo- and immunotherapy. Phase I and II trials have tested this drug in several hematopoietic and solid tumors. Pfizer has initiated Phase III trials to test PF-3512676 in combination with standard chemotherapy for non-small-cell lung cancer.     

MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4.

Toll-like receptor 4 (TLR4) is a mammalian homologue of Drosophila Toll, a leucine-rich repeat molecule that can trigger innate responses against pathogens. The TLR4 gene has recently been shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS). TLR4 may be a long-sought receptor for LPS. However, transfection of TLR4 does not confer LPS responsiveness on a recipient cell line, suggesting a requirement for an additional molecule. Here, we report that a novel molecule, MD-2, is requisite for LPS signaling of TLR4. MD-2 is physically associated with TLR4 on the cell surface and confers responsiveness to LPS. MD-2 is thus a link between TLR4 and LPS signaling. Identification of this new receptor complex has potential implications for understanding host defense, as well as pathophysiologic, mechanisms.     

TLR2和TLR4激动剂对人脐血CD34+细胞迁移能力的影响

本研究旨在探讨Toll样受体2(Toll-like receptors 2,TLR2)和TLR4激动剂对人脐血CD34+细胞迁移功能的影响及其机制.采用流式细胞术检测脐血CD34+细胞表面TLR2和TLR4的表达情况;用趋化实验和黏附实验观察PAM3CSK4(TLR2激动剂)和LPS(TLR4激动剂)对人脐血CD34+细胞的迁移活性和黏附活性的影响.结果表明:人脐血CD34+细胞表面表达TLR2(14.2±3.8)%和TLR4(19.6±4.1)%.与对照组相比,LPS可明显抑制SDF-1诱导的CD34+细胞的趋化作用(p<0.01),但LPS对CD34+细胞的黏附能力,以及PAM3CSK4对CD34+细胞的趋化和黏附能力均无明显影响.进一步研究显示,LPS对CD34+细胞表面CXCR4的表达无明显影响,但可明显抑制CD34+细胞的自发迁移作用(p<0.05).结论:TLR4的活化可显著降低SDF-1诱导CD34+细胞的趋化功能,这可能与其抑制CD34+细胞的自发迁移作用具有一定的关系.     

The delta-opioid receptor agonist SNC80 [(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-(3-methoxybenzyl)-N,N-diethylbenzamide] synergistically enhances the locomotor-activating effects of some psychomotor stimulants, but not direct dopamine agonists, in rats

The nonpeptidic delta-opioid agonist SNC80 [(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-(3-methoxybenzyl)-N,N-diethylbenzamide] produces many stimulant-like behavioral effects in rodents and monkeys, such as locomotor stimulation, generalization to cocaine in discrimination procedures, and antiparkinsonian effects. Tolerance to the locomotor-stimulating effects of SNC80 develops after a single administration of SNC80 in rats; it is not known whether cross-tolerance develops to the effects of other stimulant compounds. In the initial studies to determine whether SNC80 produced cross-tolerance to other stimulant compounds, it was discovered that amphetamine-stimulated locomotor activity was greatly enhanced in SNC80-pretreated rats. This study evaluated acute cross-tolerance between delta-opioid agonists and other locomotor-stimulating drugs. Locomotor activity was measured in male Sprague-Dawley rats implanted with radiotransmitters, and activity levels were recorded in the home cage environment. Three-hour SNC80 pretreatment produced tolerance to further delta-opioid receptor stimulation but also augmented greatly amphetamine-stimulated locomotor activity in a dose-dependent manner. Pretreatments with other delta-opioid agonists, (+)BW373U86 [(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide] and oxymorphindole (17-methyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7,2',3'-indolomorphinan), also modified amphetamine-induced activity levels. SNC80 pretreatment enhanced the stimulatory effects of the dopamine/norepinephrine transporter ligands cocaine and nomifensine (1,2,3,4-tetrahydro-2-methyl-4-phenyl-8-isoquinolinanmine maleate salt), but not the direct dopamine receptor agonists SKF81297 [R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide] and quinpirole [trans-(-)-(4alphaR)-4,4a, 5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g] quinoline monohydrochloride]. In conclusion, SNC80 enhanced the locomotor-stimulating effects of monoamine transporter ligands suggesting that delta-opioid receptor activation might alter the functional activity of monoamine transporters or presynaptic monoamine terminals.     

Characterization of a human antigen specific helper factor secreted by a T cell hybrid

Antigen specific helper factors remain incompletely characterized and somewhat controversial in part because of difficulties in generating large amounts of homogeneous helper factor. To circumvent this problem, we generated a human T cell hybrid secreting an antigen specific helper factor by fusing tetanus toxoid reactive IL-2 dependent T cells to a 6-thioguanine resistant T cell line, and screening hybrids for secreted tetanus toxoid binding molecules. A hybrid was identified that secreted a methionine containing molecule which bound tetanus toxoid, and which could induce autologous peripheral blood mononuclear cells (PBMC) to produce anti-tetanus antibodies. The helper factor stimulated anti-tetanus specifically and did not induce polyclonal immunoglobulin (Ig) synthesis. Characterization of the putative helper factor demonstrated that it carried determinants cross-reactive with HLA-DR but not Ig, bound tetanus toxoid, and had a molecular weight of 100,000-150,000. These data represent the first report of a human antigen specific helper factor generated with T cell hybrids, and demonstrate the feasibility of this approach. This approach to the generation of specific helper factors produces large amounts of helper factor, and will help in the further characterization of these molecules in human immune responses.     

Molecules from Staphylococcus aureus that bind CD14 and stimulate innate immune responses

Mammals mount a rapid inflammatory response to gram-negative bacteria by recognizing lipopolysaccharide (LPS, endotoxin). LPS binds to CD14, and the resulting LPS-CD14 complex induces synthesis of cytokines and up-regulation of adhesion molecules in a variety of cell types. Gram- positive bacteria provoke a very similar inflammatory response, but the molecules that provoke innate responses to these bacteria have not been defined. Here we show that protein-free, phenol extracts of Staphylococcus aureus contain a minor component that stimulates adhesion of neutrophils and cytokine production in monocytes and in the astrocytoma cell line, U373. Responses to this component do not absolutely require CD14, but addition of soluble CD14 enhances sensitivity of U373 cells by up to 100-fold, and blocking CD14 on monocytes decreases sensitivity nearly 1,000-fold. Deletion of residues 57-64 of CD14, which are required for responses to LPS, also eliminates CD14-dependent responses to S. aureus molecules. The stimulatory component of S. aureus binds CD14 and blocks binding of radioactive LPS. Unlike LPS, the activity of S. aureus molecules was neither enhanced by LPS binding protein nor inhibited by bactericidal/permeability increasing protein. The active factor in extracts of S. aureus is also structurally and functionally distinct from the abundant species known as lipoteichoic acid (LTA). Cell- stimulating activity fractionates differently from LTA on a reverse- phase column, pure LTA fails to stimulate cells, and LTA antagonizes the action of LPS in assays of IL-6 production. These studies suggest that mammals may use CD14 in innate responses to both gram-negative and gram-positive bacteria, and that gram-positive bacteria may contain an apparently unique, CD14-binding species that initiates cellular responses.     

Chemokine receptor expression identifies Pre-T helper (Th)1, Pre-Th2, and nonpolarized cells among human CD4+ central memory T cells

We previously reported that central-memory T cells (T(CM) cells), which express lymph node homing receptors CCR7 and CD62L, are largely devoid of effector functions but acquire characteristics of effector-memory T cells (T(EM) cells) (i.e., CCR7(-) T helper [Th]1 or Th2 cells) after stimulation with T cell receptor agonists or homeostatic cytokines. Here we show that three chemokine receptors identify functional subsets within the human CD4(+) T(CM) cell pool. T(CM) cells expressing CXCR3 secreted low amounts of interferon gamma, whereas CCR4(+) T(CM) cells produced some interleukin (IL)-4, but not IL-5. In response to IL-7 and IL-15, CXCR3(+) T(CM) and CCR4(+) T(CM) cells invariably generated fully differentiated CCR7(-) Th1 and Th2 cells, respectively, suggesting that they represent pre-Th1 and pre-Th2 cells. Conversely, CXCR5(+) T(CM) cells lacking CXCR3 and CCR4 remained nonpolarized and retained CCR7 and CD62L expression upon cytokine-driven expansion. Unlike naive cells, all memory subsets had a low T cell receptor rearrangement excision circle content, spontaneously incorporated bromodeoxyuridine ex vivo, and contained cells specific for tetanus toxoid. Conversely, recall responses to cytomegalovirus and vaccinia virus were largely restricted to CXCR3(+) T(CM) and T(EM) cells. We conclude that antigen-specific memory T cells are distributed between T(EM) cells and different subsets of T(CM) cells. Our results also explain how the quality of primary T cell responses could be maintained by T(CM) cells in the absence of antigen.     

Regulation of cytokine and toll-like receptor signaling by SOCS family genes

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through the Toll-like receptor (TLR) 4. Regulation of TLR signaling is a key step for inflammation, septic shock and innate/adaptive immunity. TLR signaling is shown to be regulated by cytokines, such as interferon-gamma (positive) and interleukin-10 and IL-4 (negative). However, molecular mechanisms of the regulation of LPS signaling by cytokines have not been clarified. Cytokine signaling is regulated by CIS/SOCS family proteins. Both SOCS1 and SOCS3 can inhibit JAK tyrosine kinase activity. We demonstrate that SOCS1 and SOCS3 play an important regulatory role in macrophages and dendritic cells (DCs) by modulating TLR signaling. SOCS1 negatively regulates not only the JAK/STAT pathway, but also the TLR-NF-kappaB pathway. SOCS3 protein was strongly induced by both IL-6 and IL-10 in the presence of LPS, but selectively inhibited IL-6 signaling. Therefore lack of SOCS3 gene in macrophages resulted in suppression of TLR signaling by hyperactivation of STAT3.