Biomedical subjects

K B Bacon

Publications and source records attributed to K B Bacon.

At least 55 records · Page 3Linked to original sources

PubMed1995

Activation of dual T cell signaling pathways by the chemokine RANTES.

The chemokine RANTES induced biphasic mobilization of Ca2+ in T cells. The initial peak, a transient increase in cytosolic Ca2+ mediated by a heterotrimeric guanine nucleotide-binding protein (G protein)--coupled pathway, was associated predominantly with chemotaxis. The second peak, Ca2+ release and sustained influx dependent on protein tyrosine kinases, was associated with a spectrum of cellular responses--Ca2+ channel opening, interleukin-2 receptor expression, cytokine release, and T cell proliferation--characteristic of T cell receptor activation. Other chemokines did not produce these responses. Thus, in addition to inducing chemotaxis, RANTES can act as an antigen-independent activator of T cells in vitro.

Benzoquinones↗

PubMed1995

Molecular cloning and functional expression of a novel CC chemokine receptor cDNA from a human basophilic cell line.

We report the cloning and characterization of a novel basophil CC chemokin receptor, K5-5, from the human immature basophilic cell line KU-812. The predicted protein sequence of K5-5 shows only 49% identity to the macrophage inflammatory protein-1 alpha/RANTES receptor (CC CKR-1) and 47% identity to monocyte chemotactic protein-1 receptor (b form), suggesting that this cDNA encodes a novel member of the CC chemokine receptor family. Analysis of K5-5 mRNA expression indicates that it is restricted to leukocyte-rich tissues. In addition, we have shown significant levels of K5-5 mRNA in human basophils, which were up-regulated by treatment with interleukin-5. The CC chemokines, Macrophage inflammatory protein-1 alpha, RANTES, and monocyte chemotactic protein-1 were able to stimulate a Ca(2+)-activated chloride channel in Xenopus laevis oocytes injected with K5-5 cRNA, whereas no signal was detected in response to monocyte chemotactic protein-2, macrophage inflammatory protein-1 beta, or the CXC chemokine, interleukin-8. Taken together, these results indicate for the first time the presence of a CC chemokine receptor on basophils, which functions as a "shared" CC chemokine receptor and may therefore be implicated in the pathogenesis of basophil-mediated allergic diseases.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and functional characterization of human lymphotactin.

We describe the isolation of a cDNA that encodes human lymphotactin (Ltn), a new class of lymphocyte-specific chemokine. Human Ltn shows similarity to some members of the C-C chemokine family but has lost the first and third cysteine residues that are characteristic of the C-C and C-X-C chemokines. Ltn is chemotactic for lymphocytes but not for monocytes, a characteristic that makes it unique among chemokines. In addition, calcium flux desensitization studies indicate that Ltn uses a unique receptor. The human Ltn gene maps to a different chromosome than do the C-C and C-X-C chemokine families. Taken together, these characteristics indicate that Ltn is the first example of a new class of human chemokines with preferential effects on lymphocytes.

Amino Acid Sequence↗

PubMed1995

Peptides from the amino-terminus of RANTES cause chemotaxis of human T-lymphocytes.

RANTES is a potent chemotactic and activating agent for a variety of leukocytes including T-lymphocytes. To identify the region of the molecule responsible for this activity, we have made overlapping 10 amino acid peptides scanning the protein. A micro-Boyden chamber chemotaxis assay showed the most efficacious peptides came from at the N-terminus. EC50 values of 8 nM (+/- 2.2 nM), 3.7 nM (+/- 2.0 nM) and 3.1 nM (+/- 2.0 nM) were calculated from dose response curves for the peptides (1-10), (3-12), and (5-14). Control peptides from other regions are not active. In THP-1 cells, none of the peptides give a Ca2+ response. The active peptides are shown to be principally chemotactic rather than chemokinetic by a checker board analysis. The results imply that the principal region of RANTES responsible for chemotaxis is located in the amino terminus.

Amino Acid Sequence↗

PubMed1995

IL-8-induced signal transduction in T lymphocytes involves receptor-mediated activation of phospholipases C and D.

We have characterized the IL-8-induced signal transduction processes in T lymphocytes. A basal level of IL-8 receptor expression was shown on mixed PBL, as identified by using phycoerythrin (PE)-coupled IL-8, and this expression was increased following IL-2 stimulation. Scatchard analysis of T cells revealed competitive binding of IL-8 with a Kd of 0.55 nM, with approximately 1200 receptors per cell, on freshly isolated T cells. After 24 h in culture following purification, reverse transcriptase PCR (RT-PCR) analyses show the mRNA for only the type B IL-8R on these cultured T lymphocytes and the cell line MOLT-4. Stimulation of T lymphocytes or T cell clones with IL-8 led to generation of inositol trisphosphate and calcium flux. In addition, when T cells were prelabeled with [3H]oleic acid, IL-8 caused a long lasting, time- and dose-related increase in [3H]phosphatidylethanol (PtE), indicating activation of phospholipase D (PLD). By contrast, this IL-8-dependent PLD activity was undetectable in IL-8-stimulated neutrophils. PLD activation appeared to be downstream of protein kinase C, because several inhibitors abrogated the increase in [3H]PtE, whereas guanosine-5'-O-(3-thiotriphosphate (GTP(gamma)S) and inositol trisphosphorothioate (IP3S3) both increased the generation of [3H]PtE. Together, these results demonstrate that the IL-8RB receptor is sufficient to mediate phospholipase C (PLC) and PLD activation in T lymphocytes, but not in neutrophils, and indicate an important difference in receptor usage and signal transduction pathways between IL-8-stimulated lymphocytes and neutrophils.

CD4-Positive T-Lymphocytes↗

PubMed1995

Mutation of Leu25 and Val27 introduces CC chemokine activity into interleukin-8.

Interleukin-8 (IL-8) is a member of the CXC branch of the chemokine superfamily and activates neutrophils but not monocytes. The related CC chemokine branch, which includes monocyte chemoattractant protein-1 (MCP-1) and RANTES are potent chemoattractants for monocytes but not neutrophils. Examination of the sequences of the CXC chemokines reveals that the highly conserved leucine, corresponding to Leu25 in IL-8, is always replaced by tyrosine in CC chemokines. There is also a high degree of conservation among the CXC chemokines of the adjacent Val27 residue, which points out from the same side of the beta-sheet as Leu25. In RANTES, Val27 is also replaced by a tyrosine. In order to investigate the role of these residues in controlling cell specificity, we have made the single mutants Leu25-->Tyr, Val27-->Tyr and the double mutant Leu25-->Tyr, Val27--> Tyr of IL-8. These proteins have been expressed in Escherichia coli and purified to homogeneity from inclusion body material. All three mutants have lower potency and efficacy in chemotaxis and calcium mobilization assays using neutrophils. The mutants also show lowered affinity to both IL-8 receptors A and B expressed recombinantly in HL-60 cells and to neutrophils in [125I]IL-8 competition assays. Additionally, the Leu25-->Tyr mutation introduces a novel monocyte chemoattractant activity into IL-8. We therefore studied the displacement of [125I]MIP-1 alpha by IL-8 Leu25-->Tyr from the CC-CKR-1 receptor. The mutant displaces MIP-1 alpha ligand with an affinity only 12-fold less than MIP-1 alpha itself. This suggests that mutations in this region of IL-8 are involved in receptor binding and activation and in the control of specificity between CC and CXC chemokines.

Amino Acid Sequence↗

PubMed1995

A fluorescent interleukin-8 receptor probe produced by targetted labelling at the amino terminus.

Interleukin-8 is the most extensively characterised member of the structurally related chemotactic and pro-inflammatory proteins collectively called chemokines. It binds to two closely related members of the seven transmembrane chemokine receptor family found on a variety of leukocyte cell types. In order to study the interaction of interleukin-8 with its receptors, and their distribution, we have produced a fluorescently labelled protein as an alternative to the radioactive 125I-interleukin-8 ligand. Interleukin-8 is naturally produced as two forms, a 72-residue polypeptide by monocytes and a 77-residue form produced by endothelial cells which has an extension of five amino acids at the amino terminal. Both forms are active at nanomolar concentrations, implying that chemical modification to the amino terminus of the 72-residue form will not destroy activity. The 72-residue interleukin-8 sequence starts with a serine residue, which can be oxidised under mild conditions to give a reactive glyoxylyl function which is then reacted with a nucleophilic fluorescein derivative. The site-specifically labelled protein was easily isolated by reverse-phase HPLC. The dissociation constant of the fluorescently labelled interleukin-8 from its receptors on neutrophils was measured by displacement of 125I-interleukin-8 and found to be 10 nM compared to 1 nM for the unmodified protein. The modified protein is highly active in in vitro bioassays using human neutrophils, giving an EC50 of 7 nM in chemotaxis and an EC50 of 0.62 nM for shape change. The binding of the fluorescent protein to neutrophils can also be measured by fluorescent automatic cell sorter (FACS) analysis, and can be competed by unlabelled interleukin-8. The amino-terminal modification of interleukin-8 has produced a reagent which is useful for the quantification of interleukin-8 receptor expression, and will also be useful in monitoring the fate of the ligand after receptor binding.

Amino Acid Sequence↗

PubMed1994

Lymphotactin: a cytokine that represents a new class of chemokine.

In this study, the cytokine-producing profile of progenitor T cells (pro-T cells) was determined. During screening of a complementary DNA library generated from activated mouse pro-T cells, a cytokine designated lymphotactin was discovered. Lymphotactin is similar to members of both the Cys-Cys and Cys-X-Cys chemokine families but lacks two of the four cysteine residues that are characteristic of the chemokines. Lymphotactin is also expressed in activated CD8+ T cells and CD4-CD8- T cell receptor alpha beta + thymocytes. It has chemotactic activity for lymphocytes but not for monocytes or neutrophils. The gene encoding lymphotactin maps to chromosome one. Taken together, these observations suggest that lymphotactin represents a novel addition to the chemokine superfamily.

Amino Acid Sequence↗

PubMed1994

Isolation of a lipid-soluble histamine release factor from human platelets.

We have isolated a histamine release factor from human platelet supernatants by heparin column chromatography, gel filtration and reversed phase HPLC. Fractions from each chromatographic step were assayed for histamine release factor activity (HRF). A peak of HRF activity was detected at a molecular mass of 60 kDa. Subsequent HPLC purification showed that the factor co-eluted with human serum albumin, but was totally extractable into the lipid phase. Comparison of biological activity with known basophil and eosinophil-activating hydroxyeicosanoids demonstrated similar activity with 5(S)-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5(S)-HPETE). The identification of this lipid soluble HRF demonstrates the novel potential role of an as yet unidentified lipid as an HRF, in the absence of priming stimuli.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and characterisation of a neutrophil chemotactic protein from porcine platelets.

In our search for novel chemoattractant factors, we have purified a heparin-binding protein from porcine platelets which is a potent chemoattractant for human neutrophils. The protein has 80 amino acids and a molecular mass of 8597.5Da as measured by electrospray mass spectrometry. It has been characterised by amino acid sequencing and shown to have highest identity to members of the human platelet basic-protein-family. Its N-terminal sequence is intermediate in length between the human connective-tissue-activating polypeptide III (CTAP-III) and neutrophil-activating polypeptide-2 (NAP-2). The porcine NAP-2/CTAP-III shows the classic CXC cysteine spacing found towards the N-terminus in the chemokine alpha family and contains the ELR motif which has been shown to be essential for neutrophil chemotaxis. We have isolated mRNA from porcine platelets and constructed a cDNA library containing 1.0 x 10(6) independent clones. Using probes based on the protein sequence we have isolated a full length-clone for this gene, with an open reading frame containing 119 amino acids. Despite overall similarity between the human and porcine proteins, the N-terminal region is almost completely different between the two species, with only two identical amino acids. The proteolytic cleavage sites required for processing of human platelet basic protein are completely missing in the porcine homologue, implying a different processing pathway or mechanism. The porcine protein is capable of agonizing certain effects of both NAP-2 and CTAP-III when incubated with human cells indicating that the same porcine protein may be involved in both processes.

Amino Acid Sequence↗

PubMed1994

Chemokines, leukocyte trafficking, and inflammation.

The relatively recent appreciation of a new class of cytokines, the chemokines, has done much to enhance our understanding of the extracellular signals involved in the movement of various populations of white blood cells. Investigation of the molecular underpinnings of chemokine function and their involvement in inflammatory processes of all kinds is beginning to yield information about the mechanisms of pathogenesis of a number of conditions, as well as providing hope for new therapeutic insights.

Animals↗

PubMed1994

Interleukin-8 and RANTES induce the adhesion of the human basophilic cell line KU-812 to human endothelial cell monolayers.

Basophils are implicated in the pathogenesis of the late-phase allergic reaction, but the mechanisms by which circulating basophils adhere to vascular endothelium and migrate to lesional sites remain unclear. In order to assess the biological similarity of the basophilic cell line KU-812 to normal human basophils, we have compared the adhesion response of this cell line and normal basophils, following challenge with interleukin-8 (IL-8) and RANTES. We demonstrate here that IL-8 and RANTES are able to stimulate the adherence of the basophilic cell line, KU-812, to cytokine-activated human umbilical vein endothelium (HUVEC). The chemokine-induced increase in adhesion was dose-related and was maximal after prior priming with IL-5. The stimulation of adhesion was partially inhibited by co-incubation with anti-CD18 and anti-CD11c antibodies and antibodies to the beta 1-integrins. In comparison, the chemokine-induced adhesion of normal human basophils was only inhibited by the beta 2-integrins. These chemokines were also able to induce the migration of KU-812 in a dose-dependent manner, but only after prior treatment with phorbol myristate acetate (PMA) or IL-5. In all cases tested, IL-8 was more potent and efficacious than RANTES. We conclude from these studies that these members of the chemokine superfamily may play an important role in the recruitment of reactive leukocytes in allergic inflammation, by stimulating their adhesion and subsequent migration from the vasculature into the inflammatory sites. However, it is apparent that KU-812 is not an adequate substitute for normal human basophils in order to investigate chemokine biology.

Base Sequence↗

PubMed1993

A fluorescence-based quantitative adhesion assay to study interactions between Entamoeba histolytica and human enterocytes. Effect of proinflammatory cytokines.

In order to study the initial events during infection of target cells by the enteric pathogen Entamoeba histolytica, we developed a quantitative adhesion assay based on the use of a human colonic cell line (CaCo-2) and biotinylated amoebae tagged with fluorescein. To prevent the strong and rapid lytic activity of Entamoeba histolytica on colonic cells, which would otherwise impede the study of the primary adhesion steps, parasites were mildly fixed, biotinylated and labelled with streptavidin-FITC. After labelled parasites have bound to enterocytes, nonadhered amoebae are removed by washing and attached parasites quantified by means of an automated fluorescence plate reader. The bioassay is simple, nonhazardous and can be completed in 1.5 h. We were able to detect ranges from 200 to 20,000 fluorescent parasites per microwell in a 96-well plate, containing approximately 10(5) colonic cells. Fluorescence intensity (arbitrary units) increased in direct relationship to the number of parasites added per well, and was not limited by the size of the culture plate (96, 24 or six wells). As an example of the value of this assay, two proinflammatory cytokines (interleukin-1, (IL-1 beta) and interferon-gamma (IFN-gamma) known to influence the adhesion properties of endothelial and epithelial cells, were used to assess their effects upon enterocyte-entamoeba binding. The increase in amoebae binding revealed by cytokine treatment to enterocytes suggests that the parasite may take advantage of inflammatory stimuli in order to increase its binding to colonic epithelium. We believe this rapid, sensitive and simple method offers the potential for large scale screening assays to study the immunobiology of this protozoal infection by analysing the mechanisms involved in the primary interactions between Entamoeba histolytica and enterocytes.

Animals↗

PubMed1993

Interleukin-8-stimulated polyphosphoinositide hydrolysis in human peripheral blood lymphocytes.

We have attempted to provide evidence for the production of inositol phosphate (IP) metabolites as an indication of specific receptor-mediated signal transduction in human peripheral blood lymphocytes (PBL) in response to interleukin-8 (IL-8). IP metabolites were measured, after loading of PBL with [3H]-D-myo-inositol, by anion exchange high-performance liquid chromatography (HPLC) and liquid scintillation counting of collected fractions. In addition, inositol-1,4,5-trisphosphate (IP3), in extracts from unlabeled cells, was measured using a specific radioligand binding assay. Compared with phytohemagglutinin (PHA), which stimulated an increase in IP metabolites and, specifically, IP3 by greater than threefold, human recombinant (hr) IL-8 (1 nmol/L) also stimulated an increase in IP metabolites, as measured by HPLC, and a greater than threefold increase in IP3. The increase in IP3 was observed as early as 15 seconds after stimulation with hrIL-8, reaching maximal levels by 30 seconds. To further assess the signal transduction mechanism involved, the protein tyrosine kinase inhibitor genistein was added to the cells 10 minutes before stimulation with hrIL-8. After preincubation of PBL with this inhibitor, the generation of IP3 in response to PHA (5 micrograms/mL) and hrIL-8 (1 nmol/L) was inhibited by 42% and 51% of control values, respectively. In contrast to the production of IP metabolites, there were only small increases in intracellular calcium in response to hrIL-8 when compared with PHA.

Calcium↗

PubMed1993

Anti-CD40 antibody stimulates the VLA-4-dependent adhesion of normal and LFA-1-deficient B cells to endothelium.

We have demonstrated that anti-CD40 antibody stimulates the heterotypic adhesion of B cells to endothelial cells. This has been shown by using normal B lymphocytes and B-cell lines in a quantitative adhesion assay. When B cells, B-cell lines and Epstein-Barr virus (EBV)-transformed B cells from a patient with leucocyte adhesion deficiency (LAD) were stimulated with anti-CD40 antibody, they were found to adhere to both untreated and interleukin-1 (IL-1)-stimulated human umbilical vein endothelial cells (HUVEC), and to the lung carcinoma line A549. To identify the adhesion receptors responsible for this anti-CD40-induced adhesion, cells were pretreated with blocking antibodies prior to assay. Our results indicate that anti-CD40-stimulated adhesion of tonsillar B cells, B-cell lines RPMI-8866, JY, and an EBV-transformed LAD-cell line were predominantly dependent on the very late antigen-4 (VLA-4)-vascular cell adhesion molecule (VCAM) interaction. Anti-CD40-induced adhesion appears to be dependent on the activation of protein tyrosine kinase and protein kinase C and on the presence of divalent cations.

Antibodies↗

PubMed1991

Characterisation of the in vitro responsiveness of lymphocyte subsets to locomotor stimuli by immunocytochemical methods.

The in vitro locomotion of lymphocyte subsets has previously been determined by use of highly purified cell populations. A method is now described in which mixed peripheral blood lymphocytes (PBL) migrate to the undersurface of polycarbonate filters in a 48 well microassay, the responding cells being characterised by alkaline phosphatase-anti-alkaline phosphatase immunocytochemistry. Recombinant interleukin (rIL)-1 alpha, zymosan activated plasma (ZAP) and rIL-8 were shown to induce concentration-related migration of mixed PBL in the 48 well assay and were therefore used as reference agonists. Total T cells, B cells, T helper/inducer and T suppressor/cytotoxic cells, as well as lymphocytes stained with the monoclonal antibodies UCHL1 and SN130, have now been quantified after migration to the undersurface of 8 microns pore size polycarbonate filters, in response to optimal concentrations of rIL-1 alpha, ZAP and rIL-8. The value of the analytical method was demonstrated by the selective responses seen. rIL-1 alpha selectively stimulated the migration of T helper/inducer and a small number of B cells without affecting T suppressor/cytotoxic cell locomotion. ZAP and rIL-8 significantly stimulated the migration of T helper/inducer, T suppressor/cytotoxic and B cells. ZAP and rIL-1 alpha also stimulated the migration of both UCHL1 and SN130 positively stained cells. This method may therefore be used to investigate the selective actions of lymphocyte locomotor stimuli on PBL sub-populations without the need to purify specific cell subsets, and to study the specificity of certain inhibitors or drugs on lymphocyte responses.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1990

Interleukin (IL)-8-induced in vitro human lymphocyte migration is inhibited by cholera and pertussis toxins and inhibitors of protein kinase C.

To further investigate the intracellular mechanisms involved in IL-8-induced human mixed peripheral blood lymphocyte (PBL) migration, the effects of pertussis toxin (PTX), cholera toxin (CTX), and protein kinase C (pkC) inhibitors were investigated. Potent inhibition of IL-8-induced PBL migration was observed following exposure of PBL to PTX and CTX (1 pM to 0.1 microM), 8-bromo cyclic adenosine monophosphate (cAMP; 1 nM to 1 microM), H7 (1 pM to 0.1 microM), sphingosine (0.1 microM to 100 microM) and the novel pkC inhibitors Ro 31-7549 and Ro 31-8220 (10 pM to 1 microM) for 10 min. Following incubation of the lymphocytes for 30 min in the presence of the direct activators of pkC, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG; 10nM to 100 microM), there was a reversal of the effects of a suboptimal dose of the specific pkC inhibitors Ro 31-7549 and Ro 31-8220. These results suggest that intracellular signals transduced during IL-8-induced in vitro PBL migration may involve pertussis and cholera toxin-sensitive G protein subunits and activation of pkC, processes which are characteristically linked to receptor binding.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Stimulation of lymphocyte migration by a novel low-molecular weight compound in normal human skin and plasma.

Normal human skin contains an extravascular population of lymphocytes which have been proposed to play a central role in immunosurveillance. The hypothesis that physiologic lymphocyte chemoattractants induce the migration of these cells has been tested by analysis of samples from normal human skin by an in vitro lymphocyte migration assay in combination with high-performance liquid chromatography (HPLC). Dilution-related lymphocyte migration was seen on assay of aqueous extracts of stratum corneum, analysis after ultrafiltration consistently demonstrating activity in less than 1-kDa fractions. Similar results were obtained with chamber fluid from abraded normal skin and with normal plasma. A novel, polar, low-molecular weight component in plasma and chamber fluid was purified to homogeneity by reverse-phase HPLC. Material from the two sources possessed identical retention times on reverse-phase HPLC and similar ultraviolet (UV) absorbance spectra (UV maximum 274 nm). The factor has been termed plasma-associated lymphocyte chemoattractant (PALC). When purified to homogeneity, PALC induced concentration-related lymphocyte migration, was extractable in lipid solvents and was not peptide in nature, as determined by mass spectrometric and amino acid analyses and proteolytic enzyme digestion. It is proposed that PALC may play a role in mediating physiologic margination, adherence and migration of lymphocytes in normal human skin.

Biological Assay↗