Biomedical subjects

Timothy J Williams

Publications and source records attributed to Timothy J Williams.

At least 19 recordsLinked to original sources

PubMed2006

The attraction of chemokines as a target for specific anti-inflammatory therapy.

Since the identification of the first chemotactic cytokines 20 years ago, the field has mushroomed, with the discovery of approximately 40 ligands, which interact with 20 different cell surface receptors. At the time of writing this review, a PubMed trawl using the word 'chemokine' will recover over 28,000 manuscripts. In this article, we will give a short history of the discovery of chemokines and provide examples of the potential for therapeutic targeting of the chemokine network in inflammatory disease.

Animals↗

PubMed2006

Evidence for autotrophy via the reverse tricarboxylic acid cycle in the marine magnetotactic coccus strain MC-1.

Strain MC-1 is a marine, microaerophilic, magnetite-producing, magnetotactic coccus phylogenetically affiliated with the alpha-Proteobacteria. Strain MC-1 grew chemolithotrophically with sulfide and thiosulfate as electron donors with HCO3-/CO2 as the sole carbon source. Experiments with cells grown microaerobically in liquid with thiosulfate and H14CO3-/14CO2 showed that all cell carbon was derived from H14CO3-/14CO2 and therefore that MC-1 is capable of chemolithoautotrophy. Cell extracts did not exhibit ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO) activity, nor were RubisCO genes found in the draft genome of MC-1. Thus, unlike other chemolithoautotrophic, magnetotactic bacteria, strain MC-1 does not appear to utilize the Calvin-Benson-Bassham cycle for autotrophy. Cell extracts did not exhibit carbon monoxide dehydrogenase activity, indicating that the acetyl-coenzyme A pathway also does not function in strain MC-1. The 13C content of whole cells of MC-1 relative to the 13C content of the inorganic carbon source (Deltadelta13C) was -11.4 per thousand. Cellular fatty acids showed enrichment of 13C relative to whole cells. Strain MC-1 cell extracts showed activities for several key enzymes of the reverse (reductive) tricarboxylic acid (rTCA) cycle including fumarate reductase, pyruvate:acceptor oxidoreductase and 2-oxoglutarate:acceptor oxidoreductase. Although ATP citrate lyase (another key enzyme of the rTCA cycle) activity was not detected in strain MC-1 using commonly used assays, cell extracts did cleave citrate, and the reaction was dependent upon the presence of ATP and coenzyme A. Thus, we infer the presence of an ATP-dependent citrate-cleaving mechanism. These results are consistent with the operation of the rTCA cycle in MC-1. Strain MC-1 appears to be the first known representative of the alpha-Proteobacteria to use the rTCA cycle for autotrophy.

Adenosine Triphosphate↗

PubMed2005

Alanine scanning mutagenesis of the chemokine receptor CCR3 reveals distinct extracellular residues involved in recognition of the eotaxin family of chemokines.

Despite considerable differences in primary structure, the chemokines eotaxin-1/CCL11, eotaxin-2/CCL24 and eotaxin-3/CCL26 signal via a single receptor, CCR3, but exhibit different potencies and efficacies. To examine receptor/ligand interactions in more detail, we performed alanine scanning mutagenesis of 21 charged residues within the extracellular loops (ECLs) of CCR3. Following transient expression in the L1.2 cell line, CCR3 mutants were assessed for their ability to be expressed at the cell surface, bind CCL11 and induce chemotactic responses to CCL11, CCL24 and CCL26. The majority of constructs were well expressed at the cell surface and bound CCL11 with low nanomolar affinity. Exceptions to this rule included the mutants E175A and E176A (ECL2) which were poorly expressed and responded weakly to all three ligands in chemotaxis assays. In contrast, the mutants K26 (amino-terminus) E179 and E180 (ECL2) responded in chemotaxis assays to CCL11 and CCL24, but not to CCL26. Mutation of residues in ECL3 was informative, with the D272A, K277A and D280A mutants exhibiting reduced chemotactic responses to two or more of the three ligands examined, despite being expressed on the cell surface at levels similar to WT CCR3. This suggests a major role for ECL3 in the recognition of all three eotaxins. In summary, distinct acidic and basic residues within CCR3 determine both receptor expression and activation by the eotaxins. Determining how these chemokines interact with their receptor at the molecular level should increase our understanding of the process of chemokine receptor activation.

Alanine↗

PubMed2005

Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors.

Mast cells are tissue-resident cells with important functions in allergy and inflammation. Pluripotential hematopoietic stem cells in the bone marrow give rise to committed mast cell progenitors that transit via the blood to tissues throughout the body, where they mature. Knowledge is limited about the factors that release mast cell progenitors from the bone marrow or recruit them to remote tissues. Mouse femoral bone marrow cells were cultured with IL-3 for 2 wk and a range of chemotactic agents were tested on the c-kit(+) population. Cells were remarkably refractory and no chemotaxis was induced by any chemokines tested. However, supernatants from activated mature mast cells induced pronounced chemotaxis, with the active principle identified as leukotriene (LT) B(4). Other activation products were inactive. LTB(4) was highly chemotactic for 2-wk-old cells, but not mature cells, correlating with a loss of mRNA for the LTB(4) receptor, BLT1. Immature cells also accumulated in vivo in response to intradermally injected LTB(4). Furthermore, LTB(4) was highly potent in attracting mast cell progenitors from freshly isolated bone marrow cell suspensions. Finally, LTB(4) was a potent chemoattractant for human cord blood-derived immature, but not mature, mast cells. These results suggest an autocrine role for LTB(4) in regulating tissue mast cell numbers.

Animals↗

PubMed2005

Identification of a 5-HT4 receptor antagonist clinical candidate through side-chain modification.

Replacement of the N-butyl side-chain of lead 5-HT4 receptor antagonist 2 with propanesulfonylpiperidinyl, morpholinyl, and piperazinyl groups led to higher affinity analogs 4-6. In vitro drug metabolism screens and cassette pharmacokinetic studies in the dog led to identification of the N-methylpiperazinyl analog (6b), which displayed pharmacokinetic, selectivity, and safety parameters sufficient for advancement to the clinic for the treatment of urinary incontinence.

Animals↗

PubMed2005

The carboxyl terminus of the chemokine receptor CCR3 contains distinct domains which regulate chemotactic signaling and receptor down-regulation in a ligand-dependent manner.

The chemokine receptor CCR3 regulates the chemotaxis of leukocytes implicated in allergic disease, such as eosinophils. Incubation of eosinophils with CCL11, CCL13 or CCL5 resulted in a rapid decrease of cell-surface CCR3 which was replicated using CCR3 transfectants. Progressive truncation of the CCR3 C terminus by 15 amino acids produced three constructs, Delta340, Delta325 and Delta310. Delta340 and Delta325 were able to bind CCL11 with affinities similar to wild-type CCR3. Delta340 transfectants exhibited enhanced migration and reduced receptor down-regulation in response to CCL11 and CCL13. Delta325 transfectants displayed chemotactic responses to CCL11 and CCL13 similar to wild-type CCR3, and had impaired down-regulation when stimulated with CCL13 but not CCL11. In contrast, neither the Delta325 nor Delta340 truncation affected chemotaxis or receptor down-regulation induced by CCL5. Delta310 transfectants bound CCL11 poorly and were biologically inactive. Inhibitors of p38 mitogen-activated protein kinase and PI3-kinase antagonized eosinophil shape change responses and chemotaxis of transfectants to CCL11 and CCL13. In contrast, shape change but not chemotaxis was sensitive to inhibition of the extracellular signal-regulated kinase kinase pathway suggesting differential regulation of the two responses. Thus, the CCR3 C terminus contains distinct domains responsible for the regulation of receptor desensitization and for coupling to chemotactic responses.

Animals↗

PubMed2004

Site-directed mutagenesis of CC chemokine receptor 1 reveals the mechanism of action of UCB 35625, a small molecule chemokine receptor antagonist.

The chemokine receptor CCR1 and its principal ligand, CCL3/MIP-1alpha, have been implicated in the pathology of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and asthma. As such, these molecules are the focus of much research with the ultimate aim of developing novel therapies. We have described previously a non-competitive small molecule antagonist of CCR1 (UCB 35625), which we hypothesized interacted with amino acids located within the receptor transmembrane (TM) helices (Sabroe, I., Peck, M. J., Jan Van Keulen, B., Jorritsma, A., Simmons, G., Clapham, P. R., Williams, T. J., and Pease, J. E. (2000) J. Biol. Chem. 275, 25985-25992). Here we describe an approach to identifying the mechanism by which the molecule antagonizes CCR1. Thirty-three point mutants of CCR1 were expressed transiently in L1.2 cells, and the cells were assessed for their capacity to migrate in response to CCL3 in the presence or absence of UCB 35625. Cells expressing the mutant constructs Y41A (TM helix 1, or TM1), Y113A (TM3), and E287A (TM7) were responsive to CCL3 but resistant to the antagonist, consistent with a role for the TM helices in CCR1 interactions with UCB 35625. Subsequent molecular modeling successfully docked the compound with CCR1 and suggests that the antagonist ligates TM1, 2, and 7 of CCR1 and severely impedes access to TM2 and TM3, a region thought to be perturbed by the chemokine amino terminus during the process of receptor activation. Insights into the mechanism of action of these compounds may facilitate the development of more potent antagonists that show promise as future therapeutic agents in the treatment of inflammatory disease.

Amino Acid Sequence↗

PubMed2004

Identification of selective basophil chemoattractants in human nasal polyps as insulin-like growth factor-1 and insulin-like growth factor-2.

In a search for novel leukocyte chemoattractants at sites of allergic inflammation, we found basophil-selective chemoattractant activity in extracts of human nasal polyps. The extracts were fractionated by reverse phase HPLC, and the resulting fractions were tested for leukocyte-stimulating activity using sensitive shape change assays. The basophil-selective activity detected was not depleted by a poxvirus CC-chemokine-binding protein affinity column. This activity was further purified by HPLC, and proteins in the bioactive fractions were analyzed by tandem electrospray mass spectrometry. Insulin-like growth factor-2 (IGF-2) was identified in these HPLC fractions, and the basophil-stimulating activity was inhibited by an anti-IGF-2-neutralizing Ab. Recombinant IGF-2 induced a substantial shape change response in basophils, but not eosinophils, neutrophils, or monocytes. IGF-2 stimulated chemokinesis of basophils, but not eosinophils or neutrophils, and synergized with eotaxin-1/CCL11 in basophil chemotaxis. IGF-2 also caused up-regulation of basophil CD11b expression and inhibited apoptosis, but did not stimulate degranulation or Ca(2+) flux. Recombinant IGF-1 exhibited similar basophil-selective effects as IGF-2, and both growth factors were detected in nasal polyp extracts by ELISA. This is the first demonstration of chemokinetic factors that increase the motility of basophils, but do not act on other granulocytes or monocytes. IGF-1 and IGF-2 could play a role in the selective recruitment of basophils in vivo.

Amino Acid Sequence↗

PubMed2004

Chemolithoautotrophy in the marine, magnetotactic bacterial strains MV-1 and MV-2.

Magnetite-producing magnetotactic bacteria collected from the oxic-anoxic transition zone of chemically stratified marine environments characterized by O2/H2S inverse double gradients, contained internal S-rich inclusions resembling elemental S globules, suggesting they oxidize reduced S compounds that could support autotrophy. Two strains of marine magnetotactic bacteria, MV-1 and MV-2, isolated from such sites grew in O2-gradient media with H2S or thiosulfate (S2O3(2-)) as electron sources and O2 as electron acceptor or anaerobically with S2O3(2-) and N2O as electron acceptor, with bicarbonate (HCO3-)/CO2 as sole C source. Cells grown with H2S contained S-rich inclusions. Cells oxidized S2O3(2-) to sulfate (SO4(2-)). Both strains grew microaerobically with formate. Neither grew microaerobically with tetrathionate (S4O6(2-)), methanol, or Fe2+ as FeS, or siderite (FeCO3). Growth with S2O3(2-) and radiolabeled 14C-HCO3- showed that cell C was derived from HCO3-/CO2. Cell-free extracts showed ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity. Southern blot analyses indicated the presence of a form II RubisCO (cbbM) but no form I (cbbL) in both strains. cbbM and cbbQ, a putative post-translational activator of RubisCO, were identified in MV-1. MV-1 and MV-2 are thus chemolithoautotrophs that use the Calvin-Benson-Bassham pathway. cbbM was also identified in Magnetospirillum magnetotacticum. Thus, magnetotactic bacteria at the oxic-anoxic transition zone of chemically stratified aquatic environments are important in C cycling and primary productivity.

Amino Acid Sequence↗

PubMed2004

The eosinophil enigma.

Eosinophils accumulate in high numbers in the lungs of asthmatic patients. These cells have the ability to induce tissue damage, a capacity that relates to their traditional role in host defense against parasitic worms. On the other hand, eosinophils produce growth factors associated with tissue repair and remodeling, notably TGF-beta1. The relationship of these activities to lung dysfunction in asthma is highly controversial, but recent observations in humans and in animal models add spice to the debate.

Animals↗

PubMed2003

Histamine induces cytoskeletal changes in human eosinophils via the H(4) receptor.

1. Histamine (0.004-2 microm) induced a concentration-dependent shape change of human eosinophils, but not of neutrophils or basophils, detected as an increase in forward scatter (FSC) in the gated autofluorescence/forward scatter (GAFS) assay. 2. The histamine-induced eosinophil shape change was completely abolished by thioperamide (10 microm), an H3/H4 receptor antagonist, but was not inhibited by pyrilamine or cimetidine (10 microm), H1 and H2 receptor antagonists, respectively. The H4 receptor agonists, clobenpropit and clozapine (0.004-2 microm), which are also H3 receptor antagonists, both induced eosinophil shape change, which was inhibited by thioperamide (10 microm). The H3/H4 receptor agonists, imetit, R-alpha-methyl histamine and N-alpha-methyl histamine (0.004-2 microm) also induced eosinophil shape change. 3. Histamine induced actin polymerisation (0.015-10 microm), intracellular calcium mobilisation (10-100 microm) and a significant upregulation of expression of the cell adhesion molecule CD11b (0.004-10 microm) in eosinophils, all of which were inhibited by thioperamide (10-100 microm). In addition, the H4 receptor agonist/H3 receptor antagonist clozapine (20 microm) stimulated a rise in intracellular calcium in eosinophils. 4. Activation of H4 receptors by histamine (1 microm) primed eosinophils for increased chemotactic responses to eotaxin, but histamine (0.1-10 microm) did not directly induce chemotaxis of eosinophils. 5. Pertussis toxin (1 microg ml-1) inhibited shape change and actin polymerisation responses induced by histamine showing that these effects are mediated by coupling to a Galphai/o G-protein. 6. This study demonstrates that human eosinophils express functional H4 receptors and may provide a novel target for allergic disease therapy.

Actins↗

PubMed2003

CCR4 blockade does not inhibit allergic airways inflammation.

The CC chemokine receptor 4 (CCR4) shows selectivity for the recruitment of memory T cell subsets, including those of the T helper cell type 2 (Th2) phenotype. In humans, CCR4+ T cells are recruited to the asthmatic lung in response to allergen challenge; however, the contribution of this pathway to allergic disease remains uncertain. We therefore investigated the role of CCR4 in allergic airways inflammation in the guinea pig. Blockade of CCR4 with a specific antibody resulted in only minor changes in numbers of CCR4+ Th cells in the bronchoalveolar lavage fluid of allergen-challenged guinea pigs and failed to inhibit the generation of eotaxin/CC chemokine ligand (CCL)11 or macrophage-derived chemokine/CCL22 or the recruitment of inflammatory leukocytes to the lung. These data suggest that although CCR4 was originally proposed as a marker of Th2 status, antigen-specific Th2 cells are recruited to the lung predominantly by other pathways. This study casts doubts on the validity of CCR4 as a therapeutic target in the treatment of asthma.

Animals↗

PubMed2003

Variations in eosinophil chemokine responses: an investigation of CCR1 and CCR3 function, expression in atopy, and identification of a functional CCR1 promoter.

We previously showed in a small group of donors that eosinophils from a subgroup of individuals responded equipotently to CC chemokine ligand (CCL)11/eotaxin and CCL3/macrophage-inflammatory protein-1alpha in assays of eosinophil shape change (CCL3/macrophage-inflammatory protein-1alpha-highly responsive (MHR) donors). In this study, we investigated the functional role of CCL3 in eosinophil responses in 73 donors. MHR donors, identified by their eosinophil shape change responses, represented approximately 19% of the donor pool. Eosinophils from these donors showed increased eosinophil CCR1 expression and also underwent CCL3-mediated chemotaxis and up-regulation of CD11b. All MHR donors gave a history of atopy-associated diseases. In a further study, we prospectively recruited 110 subjects, subdivided into nonatopics or atopics, and investigated expression of CCR1 and CCR3 on eosinophils, basophils, monocytes, and neutrophils. Eosinophil CCR1 expression was non-normally distributed in atopics, although higher CCR1 expression levels were not predictive of a diagnosis of atopy or atopic disease. We identified the CCR1 promoter and investigated its function. We found a minimal promoter within 177 bp of the transcription start site, and an upstream enhancer region that facilitated expression in leukocyte cell lines. Collectively, these data demonstrate that MHR individuals form an important subgroup that, when associated with a diagnosis of allergic disease, may require tailored therapy to modulate eosinophil recruitment. Identification of a functional CCR1 promoter will facilitate the study of possible genetic determinants underlying this potentially important clinical phenotype.

Adolescent↗

PubMed2003

Acute allergic responses induce a prompt luminal entry of airway tissue eosinophils.

Traditionally, traffic and activation of eosinophils in asthmatic airways are thought to take place during the late-phase allergic reaction. The present study tests the hypothesis that when eosinophils are present in the tissue before allergen exposure, as in chronically inflamed asthmatic airways, acute anaphylactic reactions initiate an eosinophil response. Using a guinea-pig allergic model, where eosinophilia is present at baseline conditions, the traffic of resident eosinophils was examined in vivo immediately after allergen challenge. By 2 min after challenge, eosinophils had moved up to apical epithelial positions. Within 10 min, a marked migration of eosinophils into the airway lumen was demonstrated. Along with the allergen-induced egression of eosinophils, acute luminal entry of plasma proteins and eotaxin occurred. Eosinophil egression was effectively inhibited by the antiexudative drug formoterol, whereas the proexudative drug bradykinin could in naive animals evoke a prompt luminal entry of eosinophils. In conclusion, the present study demonstrates that acute allergic reactions initiate a prompt transepithelial migration of resident eosinophils. Our data further suggest that this response in part is initiated by the plasma exudation response, which may alter the transepithelial gradient of eosinophil chemoattractants including eotaxin. We propose that prompt eosinophil response is a significant component of the acute phase of allergic reactions when occurring in airways where these cells are already present in the mucosa.

Acute Disease↗

PubMed2003

Proteoglycans are potent modulators of the biological responses of eosinophils to chemokines.

Chemokines play critical roles in governing the recruitment and activation of eosinophils at sites of allergic inflammation, particularly the asthmatic lung. However, we know little of how chemokine function is regulated post-translationally. Proteoglycans, consisting of a core protein and glycosaminoglycan (GAG) side chains, are cell surface molecules and components of the extracellular matrix that are able to bind chemokines, whilst heparin is a GAG with therapeutic value in asthma. We examined whether soluble GAG could alter the actions of chemokines in assays of eosinophil activation. Heparin inhibited intracellular calcium flux, respiratory burst and chemotactic responses of eosinophils to CCL11, but not to the chemoattractant C5a, and inhibited binding of CCL11 to CCR3. Heparin also inhibited eosinophil stimulation by CCL11, CCL24, CCL7, CCL13 and CCL5 to differing degrees, which broadly correlated with their relative affinities for heparin. Heparan sulfate and dermatan sulfate, but not chondroitin sulfate, also inhibited the actions of CCL11 and CCL13 in assays of cellular shape change and chemotaxis. Following treatment with the sulfation inhibitor chlorate or proteoglycanases, no inhibition of CCL11-induced activity was observed using either eosinophils or a CCR3-expressing transfectant cell line. This suggests that cell surface proteoglycans are not necessary for signaling via CCR3. However, the GAG context in which chemokines are expressed is likely to represent an important level of regulation of allergic inflammation.

Calcium↗

PubMed2003

Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi-step model of ligand binding and receptor activation.

CXCR3 is a chemokine receptor predominantly expressed on T lymphocytes, and binds the chemokines CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC). Here, we have investigated the role of the extracellular domains of CXCR3 in ligand selectivity and receptor activation by assessing the ligand binding and chemotactic responses of chimeric CXCR3/CXCR1 constructs. Our data reveal that the secondextracellular loop of CXCR3 is essential for receptor activation in response to all CXCR3 ligands. In contrast, the N terminus and first extracellular loop of CXCR3 play some role in CXCL10- and CXCL11-mediated activation but are dispensable for CXCL9-induced signaling. The third extracellular loop of CXCR3 is important only for CXCL9- and CXCL10-induced chemotaxis. Binding studies suggest that the CXCR3 ligands bind to distinct sites composed of multiple domains of CXCR3 and that high-affinity binding and receptor activation are disparate functions. Collectively, our data support a multi-site model for CXCR3 interactions with its agonists, in which several extracellular domains of CXCR3 contribute to ligand binding and the induction of receptor activation. The development of antagonists targeting the second extracellular loop of CXCR3 should impede receptor activation and aid the treatment of several human inflammatory disorders.

Amino Acid Sequence↗

PubMed2003

Chemokines acting via CXCR2 and CXCR4 control the release of neutrophils from the bone marrow and their return following senescence.

In this study we provide evidence that the SDF-1alpha/CXCR4 chemokine axis is involved in both the retention of neutrophils within the bone marrow and the homing of senescent neutrophils back to the bone marrow. We show that the functional responses of freshly isolated human and murine neutrophils to CXCR2 chemokines are significantly attenuated by SDF-1alpha, acting via CXCR4. As a consequence, the mobilization of neutrophils from the bone marrow in vivo by the CXCR2-chemokine, KC, was dramatically enhanced by blocking the effects of endogenous SDF-1alpha using a specific CXCR4 antagonist. As neutrophils age, they upregulate expression of CXCR4 and acquire the ability to migrate toward SDF-1alpha. We show here that these senescent CXCR4(high) neutrophils preferentially home to the bone marrow in vivo in a CXCR4-dependent manner, suggesting a previously undefined mechanism for the clearance of senescent neutrophils from the circulation.

Animals↗

PubMed2003

The effect of protein-coated contact lenses on the adhesion and viability of gram negative bacteria.

PURPOSE: Gram negative bacterial adhesion to contact lenses can cause adverse responses. During contact lens wear, components of the tear film adsorb to the contact lens. This study aimed to investigate the effect of this conditioning film on the viability of bacteria. METHODS: Bacteria adhered to contact lenses which were either unworn, worn for daily-, extended- or overnight-wear or coated with lactoferrin or lysozyme. Numbers of viable and total cells were estimated. RESULTS: The number of viable attached cells was found to be significantly lower than the total number of cells on worn (50% for strain Paer1 on daily-wear lenses) or lactoferrin-coated lenses (56% for strain Paer1). Lysozyme-coated lenses no statistically significant effect on adhesion. DISCUSSION: The conditioning film gained through wear may not inhibit bacterial adhesion, but may act adversely upon those bacteria that succeed in attaching.

Anti-Infective Agents↗