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Biomedical subjects

S Gordon

Publications and source records attributed to S Gordon.

At least 19 recordsLinked to original sources
PubMed1999

Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10.

Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-gamma (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-gamma decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-gamma stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity.

Actins↗
PubMed1999

Phagocytosis stimulates alternative glycosylation of macrosialin (mouse CD68), a macrophage-specific endosomal protein.

Macrosialin (mouse CD68), a macrophage-specific member of the lysosomal-associated membrane protein family, displays N-linked glycosylation and a heavily sialylated, mucin-like domain. We show that phagocytosis of zymosan by inflammatory peritoneal macrophages potently alters glycan processing of macrosialin in vitro. The phagocytic glycoform is not induced by other forms of endocytosis and depends on particle internalization. Zymosan uptake does not influence macrosialin protein synthesis, but increases the specific incorporation of D-[2-3H]mannose, D-[6-3H]galactose, N-acetyl-D-[1-3H]glucosamine and L-[5,6-3H]fucose by 2-15-fold. The phagocytic glycoform displays increased binding of agglutinins from peanut, Amaranthus caudatus and Galanthus nivalis, whereas binding of the sialic-acid-specific Maakia amurensis agglutinin is slightly reduced. Digestion by N-Glycanase abolishes the incorporation of [3H]mannose label and Galanthus nivalis agglutinin binding activity, but preserves the incorporation of galactose and N-acetylglucosamine and specific lectin binding. We also show that phagocytosis increases the complexity and length of O-linked chains. The data presented highlight the importance of differential glycosylation in the biology of macrosialin, phagosomes and macrophages in general.

Animals↗
PubMed1999

Previous uptake of apoptotic neutrophils or ligation of integrin receptors downmodulates the ability of macrophages to ingest apoptotic neutrophils.

Clearance of apoptotic neutrophils (polymorphonuclear leukocyte [PMN]) by macrophages is thought to play a crucial role in resolution of acute inflammation. There is increasing evidence that ingestion of apoptotic cells modulates macrophage behavior. We therefore performed experiments to determine whether ingestion of apoptotic PMN modulated the uptake process itself. Rat bone marrow-derived macrophages (BMDM) ingested apoptotic PMN by a process that was enhanced by tumor necrosis factor (TNF) and attenuated by interferon (IFN)-gamma, interleukin (IL)-4, and IL-10. It was inhibitable by the tetrapeptide arg-gly-gln-ser (RGDS), therefore implicating the alphavbeta3/CD36/thrombospondin pathway. Interaction of apoptotic PMN with BMDM for 30 minutes, 48 hours before rechallenge reduced uptake of apoptotic PMN by 50% compared with previously unchallenged BMDM. Blocking initial uptake with RGDS abrogated the effect of preexposure. Comparable and sustained attenuation of uptake was obtained by ligating alphavbeta3 with the monoclonal antibody (MoAb), F11, after a delay of more than 90 minutes, whereas MoAbs to CD25 and CD45 had no effect. Ligation of alpha6beta1 and alpha1beta2, integrins not previously implicated in the engulfment of apoptotic cells also decreased uptake with similar kinetics to F11. Therefore, apoptotic PMN regulate their own uptake through an integrin-dependent process, which can be reproduced by ligation of other integrins expressed by macrophages.

Animals↗
PubMed1999

Regulation and functional involvement of macrophage scavenger receptor MARCO in clearance of bacteria in vivo.

The scavenger receptors expressed by macrophages are thought to play an important role in the immune response against bacteria by mediating binding and phagocytosis. A novel member of the class A scavenger receptor family, macrophage receptor with collagenous structure (MARCO), has recently been identified. In this study we have generated a panel of mAbs with specificities for different domains of this receptor. Two of those reacting with the C-terminal cysteine-rich domain block ligand binding of MARCO. The in vivo expression of this murine receptor is normally restricted to distinct populations of macrophages in the spleen and lymph nodes. During bacillus Calmette-Guérin (BCG) infection, during bacterial sepsis, or after the injection of purified LPS, however, the expression of MARCO is rapidly induced on macrophages in other tissues, including Kupffer cells in the liver. Using the mouse macrophage cell line J774.2, it was shown that LPS stimulation up-regulates surface expression of MARCO in a dose- and time-dependent fashion. The proinflammatory cytokines IL-1, IL-6, TNF-alpha, and IFN-gamma had little or no effect. Using inhibitory mAbs, the relevance of MARCO for the clearance of circulating bacteria in vivo was determined. Although the overall elimination of live Escherichia coli and Staphylococcus aureus from the blood did not appear to be affected by treatment with these Abs, the capturing of heat-killed bacteria by macrophages in the marginal zone areas of the spleen was clearly inhibited. This study suggests a role for MARCO in the host antibacterial defense.

Animals↗
PubMed1999

Exposure-response relationships for work-related sensitization in workers exposed to rat urinary allergens: results from a pooled study.

BACKGROUND: Recent studies in a few industries have shown that the likelihood of IgE-mediated sensitization increases with increasing exposure. The shape of the exposure-response relationships and modification by age, sex, and smoking habit has hardly been studied. OBJECTIVE: The purpose of this study was to determine exposure sensitization relationships for rat sensitization and to evaluate the influence of atopy, smoking habits, and sex. METHODS: Data from 3 cross-sectional studies in The Netherlands, the United Kingdom, and Sweden were used and involved 1062 animal laboratory workers. Selection criteria were harmonized, and this resulted in a study population of 650 animal laboratory workers (60.6% female) with less than 4 years of exposure. Air allergen levels were assessed previously and converted on the basis of an interlaboratory allergen analysis comparison. Available sera were analyzed for the presence of specific antibodies against common allergens (house dust mite, cat, dog, and grass and birch pollen) and work-related allergens (rat and mouse urinary proteins). Questionnaire items on work-related respiratory symptoms, hours worked with rats per week, job performed, smoking habits, and sex were used in this analysis RESULTS: The prevalence of work-related sensitization to rat urinary allergens (IgE >0.7 KU/L) was 9.7 % (n = 63). Thirty-six of the sensitized workers had work-related symptoms (asthma or rhinitis). Two hundred forty-eight workers (38.2%) were atopic (defined as specific IgE to 1 of the common allergens). The sensitization rate increased with increasing air allergen exposure. Atopic workers exposed to low levels of allergen had a more than 3-fold increased sensitization risk compared with nonexposed atopic workers. For atopic subjects, the risk increased little with increasing exposure, whereas for nonatopic subjects, a steadily increasing risk was observed. Smoking and sex did not modify the sensitization risk. CONCLUSION: Rat urinary allergen-sensitization risk increased with increasing exposure intensity. Workers who were atopic had a clearly elevated sensitization risk at low allergen exposure levels.

Allergens↗
PubMed1999

Macrophage-restricted molecules: role in differentiation and activation.

Membrane antigens serve as excellent markers of murine macrophage (MO) differentiation in vivo and in vitro, and have yielded insights into novel MO functions in innate and acquired immunity. We review briefly the use of a range of monoclonal antibodies against MO which show restricted expression: F4/80, macrosialin and sialoadhesin. Differential display PCR strategies have made it possible to identify a novel MO-restricted membrane molecule, a murine MO C-type lectin (mMCL). The isolation of functionally active ab against known receptors such as the scavenger receptor, Class A (SR-A) has provided a key reagent to characterise the role of this molecule in adhesion, endocytosis, phagocytosis and in the regulation of cellular immunity. Studies in SR-A ko mice challenged with BCG and LPS show that the SR-A molecule helps to limit excessive production of cytokines such as TNFalpha by MO that have been activated by infection, and thus protects the host against septic shock.

Animals↗
PubMed1999

Potential role of the mannose receptor in antigen transport.

Potential endogenous ligands for the cysteine rich domain of the murine mannose receptor (MR) have been detected in marginal zone metallophilic macrophages in spleen and subcapsular sinus macrophages in lymph nodes of naive mice by immunohistochemistry using a Fc chimeric protein. Additional labelling was observed in follicular dendritic cells and migratory dendritic cells in immunised animals. Based on this labelling pattern and the identification of a soluble form of the MR in macrophage-conditioned media and mouse serum, we propose a novel role for this receptor in antigen transport.

Animals↗
PubMed1999

Class A scavenger receptors and the phagocytosis of apoptotic cells.

Apoptotic cells are rarely seen in situ because of rapid clearance by phagocytes. A number of receptors have been implicated in the recognition and ingestion of dying cells. Class A scavenger receptors (SRs) are multi-domained membrane glycoproteins that can endocytose modified lipoproteins and bind a wide range of ligands. There is growing evidence that they contribute to several biological processes. We present data that suggest class A SRs are involved in the phagocytosis of apoptotic cells.

Animals↗
PubMed1999

Comparison of methods to assess airborne rat and mouse allergen levels. I. Analysis of air samples.

Airborne laboratory-animal allergens can be measured by several methods, but little is known about the effects of important differences in methodology. Therefore, methods used in research projects in The Netherlands, the UK, and Sweden were compared. Seventy-four sets of three parallel inhalable dust samples were taken by a single operator in animal facilities in the three countries, and analyzed in parallel by the three institutes for rat and mouse urinary allergen. Rat-allergen levels measured by RAST inhibition (UK) were 3000 and 1700 times higher than levels measured by enzyme immunoassay (EIA)-sandwich methods with polyclonal rabbit (The Netherlands) or monoclonal mouse (Sweden) antibodies, while the difference between the two EIA-sandwich methods was much smaller: a factor of 2.2. For mouse allergen, an inhibition radioimmunoassay (RIA) with rabbit antimouse antibodies (UK) gave 4.6 and 5.9 times higher concentrations than sandwich EIAs with rabbit polyclonal antibodies (Sweden and The Netherlands), while the difference between the two sandwich EIAs was, on average, 1.6-fold. Thus, although levels of rat and mouse aeroallergens are significantly correlated, the assay type gives large differences in absolute concentrations, and interlaboratory technical differences affect even the same assay type. Conversion factors can aid comparison between studies, and, in the long term, assay standardization is desirable.

Air Pollutants↗
PubMed1999

Comparison of methods to assess airborne rat and mouse allergen levels. II. Factors influencing antigen detection.

Potential factors influencing antigen detection in immunoassays for measuring rat or mouse aeroallergen (i.e., assay setup, antigen specificities, standard extracts used, and antigen decay) were investigated in a three-country study (the UK, The Netherlands, Sweden). An inhibition enzyme immunoassay (EIA) setup gave nominal rat urinary allergen (RUA) sample values seven times higher than a sandwich EIA setup utilizing identical antibodies and standards. In immunoblotting experiments, pooled patient serum and polyclonal rabbit antibodies partly detected different rat antigens; monoclonal antibody specificity could not be determined. Immunoblot detection of mouse urinary antigens (MUA) by the polyclonal rabbit antibodies from all laboratories was similar. In both the RUA and the MUA assays, urinary antigen standards were detected with similar potency, except purified Rat n 1, which was an inefficient inhibitor in the RUA RAST inhibition. In the sandwich EIA RUA assays, a rat room-dust extract was detected with 700800-fold less sensitivity than rat urine, whereas in the RAST RUA assay, dust inhibited equally with rat urine. Simulated decay did not decrease the potency of urinary antigen in any assay. Thus, assay setup and choice of detection antibodies strongly influence the nominal allergen levels. We recommend the use of standardized and characterized antibodies and standard extracts in sandwich EIAs to measure airborne rodent urinary allergens.

Air Pollutants↗
PubMed1999

Lymphocyte subpopulations in lymphoid organs of rats after acute resistance exercise.

PURPOSE: The ability of aerobic exercise to change lymphocyte subpopulation distributions is well documented; much less is known about resistance exercise. The purpose of this experiment was to determine the effects of an acute bout of resistance exercise on lymphocyte subpopulations in primary and secondary lymphoid compartments. METHODS: Male rats were operantly conditioned to climb a ladder while carrying weights that were progressively increased to equal body weight. During the acute session, rats performed repetitive climbs until exhaustion. Thymus, spleen, blood, and axial and inguinal lymph nodes were removed; leukocytes were isolated and incubated with monoclonal antibodies against differentiation markers, activation antigens, and adhesion molecules. RESULTS: Exercised versus control rats had greater numbers of leukocytes in the thymus, axial, and inguinal nodes but not in the blood or spleen. The percentage of CD4+ cells increased after exercise in the thymus, spleen, and blood. The percentages of cells expressing the integrin LFA-1beta were elevated in all the tissues except inguinal lymph nodes. In addition, more leukocytes from exercised than nonexercised rats expressed detectable numbers of activation markers, IL-2 receptor-alpha and MHC class II molecules; however, as indicated by proliferating cell nuclear antigen analysis, the cells were not actively dividing at the time of assay. CONCLUSIONS: Based on these and published data, it appears that a single bout of resistance exercise can affect lymphoid cell subpopulations probably by inducing changes in leukocyte trafficking.

Animals↗
PubMed1999

Comparative analysis and serovar-specific identification of multiple-banded antigen genes of Ureaplasma urealyticum biovar 1.

Ureaplasma urealyticum is a causative agent of nongonococcal urethritis and is implicated in the pathogenesis of several other diseases. The species is divided into 14 serovars and two biovars, of which biovar 1 is most commonly isolated from clinical specimens. Reported associations between individual serovars and diseases have been difficult to confirm because of practical difficulties with serotyping. The multiple-banded antigen (MBA) is the predominant U. urealyticum antigen recognized during infections in humans and probably has a significant role in virulence. The 5' end of the MBA gene is relatively conserved but contains biovar, and possibly serovar, specificity. The 5' ends of the MBA genes of standard strains of U. urealyticum biovar 1, consisting of serovars 1, 3, 6, and 14, were amplified, cloned into pUC19, and sequenced to identify serovar-specific differences. The 5' end of the MBA gene sequence of serovar 3 was identical with the previously published sequence and differed by only three bases from that of serovar 14. Significant differences between the MBA gene sequences allowed biovar 1 to be divided into two subgroups, containing serovars 3/14 and serovars 1 and 6, respectively, using primers UMS-125-UMA269 and UMS-125-UMA269'. Serovars 1 and 6 were distinguished by restriction enzyme analysis of the amplicon and/or by PCR specific for serovar 6. These methods were used to identify and type U. urealyticum in 185 (46.3%) of 400 genital specimens from women. Biovar 1 was detected in 89.2% and biovar 2 in 18.3% of positive specimens. Of 165 specimens containing U. urealyticum biovar 1, 22.2% contained more than one serovar and 46.7, 46.1, and 25.5% contained serovars 1, 3/14, and 6, respectively. U. urealyticum was found in a significantly higher proportion of pregnant women than in sex workers and other women attending a sexually transmissible diseases clinic (P < 0.01). The methods described are relatively rapid, practicable, and specific for serotyping isolates and for direct detection and identification of individual serovars in clinical specimens containing U. urealyticum biovar 1.

Antigens, Bacterial↗
PubMed1999

Analysis of macrophage scavenger receptor (SR-A) expression in human aortic atherosclerotic lesions.

The class A scavenger receptors (SR-As) are trimeric, integral membrane glycoproteins that exhibit unusually broad ligand-binding properties. A number of studies have suggested that these receptors may play an important role in host defense and in many macrophage-associated pathological processes, including atherosclerosis and Alzheimer's disease. The study of the expression and function of these receptors in human disease has been hampered by the lack of suitable antibodies recognizing human SR-A. This has generated questions regarding the nature of receptors responsible for scavenger receptor activity detected in a variety of cell types, including monocytes, macrophages, smooth muscle cells, and endothelial cells. To address these questions, we have produced high-titer antisera recognizing human SR-A by using mice deficient for SR-A (SR-A -/-). We show that SR-A -/- mice produce a significantly higher-titer immune response than do wild-type (SR-A +/+) littermates, with antisera of the former having a broad species reactivity and recognizing SR-A from humans, mice, and rabbits. The antisera recognize both type I and II SR-A in a wide range of immunological techniques. Using these antisera we show that the expression of SR-A protein is induced during monocyte to macrophage differentiation and that SR-A mediates 80% of the uptake of acetylated low density lipoprotein by human monocyte-derived macrophages. We also establish that human SR-A is expressed by tissue macrophages in liver and lung and by macrophage-derived foam cells within aortic atherosclerotic lesions, with little detectable expression by smooth muscle cells or aortic endothelium.

Actins↗
PubMed1998

Functional comparison of the murine macrosialin and human CD68 promoters in macrophage and nonmacrophage cell lines.

The glycoprotein macrosialin is expressed specifically in murine monocytes and macrophages. In the murine genome the macrosialin gene lies 877 bp 3' of the ubiquitously expressed elongation initiation factor 4A1 (eIF-4AI) gene on chromosome 11. The macrosialin gene promoter directs high-level expression in transiently transfected murine macrophage cell lines, but significant levels of expression are seen in nonmacrophage cell lines. The first intron of the gene encoding the human homologue of macrosialin, CD68, directs macrophage-specific expression when cloned downstream of the human CD68 gene promoter. Despite sharing extensive sequence homology with the human CD68 gene first intron, the murine macrosialin gene first intron does not act as a macrophage-specific enhancer when cloned downstream of the human CD68 or murine macrosialin promoters. We conclude that the genetic elements that are responsible for macrophage-specific gene expression are organized differently in the murine and human CD68 genes.

Animals↗
PubMed1998

A functional soluble form of the murine mannose receptor is produced by macrophages in vitro and is present in mouse serum.

A soluble form of the mannose receptor (sMR) has been found in conditioned medium of primary macrophages in vitro and in mouse serum. sMR was released as a single species, had a smaller size than the cell-associated form, and accumulated in macrophage-conditioned medium, in a cytokine-regulated manner, to levels comparable with those found for cell-associated mannose receptor. Pulse-chase experiments showed that sMR production in culture occurred by constitutive cleavage of pre-existing full-length protein. A binding assay was developed to determine the sugar specificity of sMR and its ability to interact with pathogens and particulate antigens (i.e. Candida albicans and zymosan). Protease inhibitor studies suggested that sMR was produced by cleavage of an intact mannose receptor by a matrix metalloprotease or ADAM metalloprotease. A role for sMR in the immune response is proposed based on its binding properties, regulation by cytokines, and the previous discovery of putative ligands for the cysteine-rich domain of the mannose receptor in lymph nodes and spleen.

Amino Acid Sequence↗
PubMed1998

Studies of dehydroepiandrosterone (DHEA) with the human estrogen receptor in yeast.

Dehydroepiandrosterone (DHEA) is a C19 adrenal steroid synthesized in the human adrenal cortex and serving as a biosynthetic precursor to testosterone and 17beta-estradiol. Despite the fact that it is one of the most abundant steroid hormones in circulation, the physiological role of DHEA in humans remains unclear. The action of DHEA itself, such as its interactions with receptors and nuclear transcription factors, is not well understood, and a specific DHEA receptor has yet to be identified. Although the activity of DHEA can be due to its metabolism into androgens and estrogens, DHEA has been shown to interact with the androgen receptor and the estrogen receptor (ER) in vitro. We demonstrate in this study that DHEA (3beta-Hydroxy-5alpha-androstan-17-one) inhibits 17beta-estradiol (E2) binding to its receptor in vivo in yeast. DHEA stimulates human ER dimerization in yeast, as determined by ER fusion protein interactions, GAL4 reconstitution and subsequent measurement of increased beta-galactosidase activity. DHEA causes an increase in estrogen response element-dependent beta-galactosidase activity, demonstrating that the ER dimer induced by DHEA is transcriptionally active, but at a concentration of DHEA about 1000 times greater than E2. Inclusion of the nuclear receptor co-activator RIP140 in the yeast enhances ER transactivation by DHEA or E2 in a ligand-dependent manner; moreover, only in the presence of RIP140 is DHEA able to stimulate beta-galactosidase activity to levels similar to those achieved by E2. Ligand-receptor interaction for other C19-steroids was also examined. While 5-androstene-3beta, 17beta-diol (ADIOL) displayed estrogenic activity in this system, 4-androstene-17-dione (androstenedione) and 4-androstene-17beta-ol,3-one (testosterone) did not. We have investigated whether DHEA can interact with the human ER in vivo. Our findings demonstrate a mechanism by which DHEA interacts directly with estrogen signaling systems; however, because DHEA is several orders of magnitude less potent than E2 in this system, we conclude that it essentially is not an estrogen agonist.

Dehydroepiandrosterone↗