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K L McCoy

Publications and source records attributed to K L McCoy.

At least 19 recordsLinked to original sources
PubMed2000

Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB(2) receptor.

Cannabinoids have immunomodulatory as well as psychoactive effects. Because the central cannabinoid receptor (cannabinoid CB(1) receptor) is highly expressed in many neuronal tissues and the peripheral cannabinoid receptor (cannabinoid CB(2) receptor) is highly expressed in immune cells, it has been suggested that the central nervous system effects of cannabinoids are mediated by cannabinoid CB(1) receptors and that the immune effects are mediated by cannabinoid CB(2) receptors. To test this hypothesis, we have generated the first mouse strain with a targeted mutation in the cannabinoid CB(2) receptor gene. Binding studies using the highly specific synthetic cannabinoid receptor agonist (-)-cis-3-¿2-Hydroxy-4-(1, 1-dimethylheptyl)phenyl-trans-4-(3-hydroxypropyl)cyclohexanol (¿3HCP 55,940) revealed no residual cannabinoid binding sites in the spleen of the cannabinoid CB(2) receptor knockout mice, while binding in the central nervous system was unchanged. Cannabinoid CB(2) receptor knockout mice, which appear healthy, are fertile and care for their offspring. Fluorescence activated cell sorting (FACS) analysis showed no differences in immune cell populations between cannabinoid CB(2) receptor knockout and wildtype mice. We investigated the immunomodulatory effects of cannabinoids in cannabinoid CB(2) receptor deficient mice using a T cell co-stimulation assay. Delta(9)Tetrahydrocannabinol inhibits helper T cell activation through macrophages derived from wild type, but not from knockout mice, thus indicating that this effect is mediated by the cannabinoid CB(2) receptor. In contrast, central nervous system effects of cannabinoids were not altered in these mice. Our results suggest that cannabinoid CB(2) receptor-specific ligands may be clinically useful in the modulation of macrophage immune function while exhibiting no central nervous system activity. Furthermore, we conclude that the cannabinoid CB(2) receptor knockout mouse is a useful animal model in which to study the role of the cannabinoid system in immunoregulation.

Animals↗
PubMed2000

Delta(9)-tetrahydrocannabinol selectively increases aspartyl cathepsin D proteolytic activity and impairs lysozyme processing by macrophages.

Delta(9)-tetrahydrocannabinol (THC) causes an antigen-dependent defect in the ability of macrophages to activate helper T cells, and this drug-induced impairment is mediated through the peripheral CB2 receptor. Various requirements for the processing of the antigen, lysozyme, were examined to determine where along the pathway THC exerts its influence. A THC-exposed macrophage hybridoma inefficiently stimulated interleukin-2 secretion by a helper T cell hybridoma in response to native lysozyme and its reduced form, suggesting that disulfide bond reduction was unaffected. Cell surface expression of major histocompatibility complex class II molecules was normal on THC-exposed macrophages. The drug-exposed macrophages also competently presented a lysozyme peptide to the T cells, indicating that the class II molecules were functional. The proteolytic activity of two thiol cathepsins was unaltered, but aspartyl cathepsin D activity was significantly increased in THC-exposed macrophages. Thus, selective up-regulation of aspartyl cathepsin activity accompanied the deficiency in lysozyme processing and may contribute, at least in part, to the antigen-dependent processing defect in THC-exposed macrophages.

Animals↗
PubMed1999

Use of venous duplex scans to evaluate symptoms of deep vein thrombosis: an analysis of ultrasound usage by various medical specialties.

In today's environment of cost-containment and utilization management, duplex ultrasound is often overused to evaluate symptoms of deep venous thrombosis/thrombophlebitis (DVT), reflecting the low diagnostic yield of such studies. We investigated the use of venous duplex scans by various medical specialties to determine whether a tendency exists to overuse this diagnostic tool by one specialty compared with others and to assess the cost-effectiveness of ordering this test for acute venous disease changes. We retrospectively reviewed the results of venous duplex ultrasound studies for 330 consecutive patients with suspected DVT for a 1-year period. Our analysis showed that 51 of 330 (16%) of all duplex scans ordered in our institution were positive for DVT. Internists, as a group, ordered 185 studies of which 26 were positive (14%). The surgeons' requests for duplex studies resulted in 23 of 137 (17%) confirmed positive studies. The overall positive examination rate was 16 per cent, which is not only suboptimal as a diagnostic tool, but also cumbersome with regard to health care cost-containment.

Cost-Benefit Analysis↗
PubMed1999

Cannabinoid inhibition of the processing of intact lysozyme by macrophages: evidence for CB2 receptor participation.

Delta9-tetrahydrocannabinol (THC) impairs multiple immunological functions. The ability of a macrophage hybridoma to function as an antigen-presenting cell was examined by the stimulation of a soluble protein antigen-specific helper T cell hybridoma to secrete interleukin-2. THC exposure significantly reduced the T cell response to the native form of the antigen after a 24-h pretreatment of the macrophages with nanomolar drug concentrations. However, THC did not affect interleukin-2 production when the macrophages presented a synthetic peptide of the antigen to the T cells, suggesting that the drug may interfere with antigen processing, not peptide presentation. Cannabinoid inhibition of the T cell response to the native antigen was stereoselective consistent with the involvement of a cannabinoid (CB) receptor. Bioactive CP-55,940 diminished T cell activation, whereas the inactive stereoisomer CP-56,667 did not. The macrophage hybridoma expressed mRNA for the CB2 but not the CB1 receptor whereas the T cells expressed an extremely low level of mRNA for the CB2 receptor. The CB1-selective antagonist SR141716A did not reverse the suppression caused by THC, demonstrating that the CB1 receptor was not responsible for the drug's inhibitory effect. In contrast, the CB2-selective antagonist SR144528 completely blocked THC's suppression of the T cell response, implicating the participation of the CB2 receptor. These findings suggest that the CB2 receptor may be involved in CB inhibition of antigen processing by macrophages in this system.

Animals↗
PubMed1998

Gallium arsenide modulates proteolytic cathepsin activities and antigen processing by macrophages.

Gallium arsenide (GaAs) is a semiconductor utilized in the electronics industry. Chemical exposure of animals causes a local inflammatory reaction, but systemic immunosuppression. Mice were administered i.p. 200 mg/kg GaAs crystals or latex beads, or vehicle. Five days after exposure, splenic macrophages were defective, whereas thioglycolate-elicited peritoneal macrophages (PEC) were more efficient in processing the Ag, pigeon cytochrome c, than vehicle control macrophages. Various aspects of the MHC class II Ag-processing pathway were examined. Both macrophage populations normally presented a peptide fragment to the CD4+ T cells. Surface MHC class II expression on the PEC was up-regulated, but splenic cells had normal MHC class II expression. PEC had elevated levels of glutathione and cysteine, major physiologic reducing thiols. However, the cysteine content of splenic macrophages was diminished. Proteolytic activities of aspartyl cathepsin D, and thiol cathepsins B and L were decreased significantly in splenic macrophages. On the other hand, thiol cathepsin activities were increased selectively in PEC. Latex bead-exposed PEC were not more potent APC, and their thiol cathepsin activities were unchanged, indicating that phagocytosis and nonspecific irritation were not responsible. The phenotype of PEC directly exposed to GaAs mirrored cytokine-activated macrophages, in contrast to splenic macrophages from a distant site. Therefore, GaAs exposure differentially modulated cathepsin activities in splenic macrophages and PEC, which correlated with their Ag-processing efficiency. Perhaps such distinct alterations may contribute to the local inflammation and systemic immunotoxicity caused by chemical exposure.

Adjuvants, Immunologic↗
PubMed1998

Gallium arsenide differentially affects processing of phagolysosomal targeted antigen by macrophages.

Gallium arsenide, a semiconductor utilized in the electronics industry, causes immunosuppression in animals. The chemical's effect on macrophages to process antigen for activating pigeon cytochrome-specific helper T cell hybridoma was investigated. Mice were administered 200 mg/kg gallium arsenide or vehicle intraperitoneally. Five-day exposure suppressed processing by splenic macrophages but augmented processing by thioglycollate-elicited and resident peritoneal macrophages. Cytochrome coupled to latex beads was targeted to phagolysosomes to examine processing in lysosomes. Cytochrome beads required phagocytosis for processing and were located in phagolysosomes. Gallium arsenide did not alter the phagocytic ability of macrophages. Peritoneal macrophages normally processed the targeted antigen, indicating that gallium arsenide influenced compartment(s) preceding lysosomes. However, the processing efficiency of exposed splenic macrophages depended on the size of particulate cytochrome, suggesting that processing varied in phagolysosomes of different sizes. Gallium arsenide impacted different intracellular compartments in these macrophages, perhaps contributing to systemic immunotoxicity and local inflammation caused by exposure.

Animals↗
PubMed1998

Direct exposure to gallium arsenide upregulates costimulatory activity of murine macrophages.

Gallium arsenide (GaAs) is an intermetallic semiconductor compound used in the electronics industry. Acute exposure of animals to GaAs systemically suppresses several immune functions while paradoxically causing inflammation at the exposure site. We investigated the effect of GaAs on costimulatory activity of murine peritoneal macrophages, 5 days after ip exposure. Costimulation by macrophages was determined by activation of CD4(+) helper T cell hybridomas to secrete interleukin-2 in the presence of immobilized monoclonal anti-CD3 antibody. Both peritoneal exudate cells (PEC) and resident peritoneal cells exposed to GaAs provided greater costimulation to the T cells than vehicle control cells. Resident peritoneal cells exposed to GaAs were also more efficient than latex bead-exposed cells, indicating that phagocytosis alone did not cause the GaAs effect. Double immunofluorescence staining and flow cytometric analysis revealed that GaAs-exposed PEC had increased cell surface expression of costimulatory B7-1 and B7-2 molecules and intracellular adhesion molecule-1 (ICAM-1) compared to controls. In addition to these molecules, resident peritoneal macrophages exposed to GaAs also expressed significantly higher levels of heat-stable antigen (HSA). Monoclonal antibodies specific for these costimulatory molecules significantly inhibited T cell activation, demonstrating that the molecules on GaAs-exposed cells were functional. In contrast, GaAs did not upregulate costimulatory molecules on splenic macrophages. These findings suggest that direct GaAs exposure improves macrophage costimulatory activity, possibly by activating the cells, which may contribute to respiratory inflammation caused by inhalation of GaAs particles.

Animals↗
PubMed1998

Delta9-tetrahydrocannabinol suppresses macrophage costimulation by decreasing heat-stable antigen expression.

Delta9-tetrahydrocannabinol (THC) suppresses several immunologic functions of macrophages. The costimulatory activity of a THC-exposed macrophage hybridoma was investigated by its ability to elicit interleukin-2 secretion by a helper T cell hybridoma activated with immobilized monoclonal anti-CD3 antibody. THC added at culture initiation inhibited the T cell response in a dose-dependent manner. When the macrophages were fixed with paraformaldehyde before culture, THC had no effect on T cell stimulation. However, macrophages, which were preincubated with THC and then fixed, were impaired in delivering costimulatory signals to T cells cultured without THC. The drug's inhibitory effect on macrophage costimulatory activity was reversible. THC exposure also decreased macrophage expression of heat-stable antigen (HSA). Antibody blocking experiments showed that HSA expressed on the macrophages provided an important costimulatory signal, whereas B7-1 and B7-2 molecules had a minor role. Treatment of the macrophages with phosphatidylinositol-specific phospholipase C cleaved HSA, but not the transmembrane B7 molecules, from the cell surface. Similar to THC, enzyme treatment significantly diminished macrophage costimulatory activity, which was also reversible. After drug or enzyme removal, HSA expression returned to the control level by 4 h. Therefore, THC suppresses macrophage costimulatory activity by diminishing cell surface expression of HSA.

Antibodies, Monoclonal↗
PubMed1996

Intracellular location of cysteine transport activity correlates with productive processing of antigen disulfide.

Activation of CD4+ T cells requires processing of exogenous protein antigens by antigen-presenting cells (APC). A macrophage hybridoma and B cell lymphoma were comparable in their ability to process hen egg lysozyme (HEL), which involves reduction of its disulfide bonds. The intracellular levels of cysteine and glutathione, major physiological thiols, based on protein content were similar within these cell lines. In addition, the cysteine transport pathway in viable cells was assessed by 35S-cystine uptake. For macrophages, the majority of the radioactivity resided in high density subcellular fractions of Percoll gradients that comigrated with lysosomal beta-galactosidase (beta-gal). Besides the lysosomes, low density fractions cosedimenting with endosomes incorporated the radiolabel in the B cells. Both peaks of radioactivity disappeared when the B cells were incubated with unlabeled carboxymethyl-cysteine (CM-cysteine), a specific competitor of the plasma membrane CG transport system. The distinct gradient profiles of radiolabel uptake in the cells correlated with a difference in their capacity to process the transferrin-lysozyme conjugate (TF-HEL). TF-HEL was significantly more stimulatory than HEL in inducing a HEL-specific T cell response with the B cells as the APC. However, the potencies of TF-HEL and HEL were similar when the macrophages were the APC. Thus, the intracellular location of cysteine transport activity may be cell lineage-dependent, and its presence may, in part, determine whether an organelle is a productive site of processing antigens with disulfide bonds that is necessary for CD4+ cell activation.

Antigen Presentation↗
PubMed1996

Defective antigen processing correlates with a low level of intracellular glutathione.

Previously, we reported that Chinese hamster ovary (CHO) cells transfected with murine mouse major histocompatibility complex class II genes, exhibit a unique antigen (Ag) processing defect whereby these cells are impaired in processing only Ag with disulfide bonds. Here, we examined various aspects of the intracellular reducing environment in the CHO cells to understand the underlying mechanism causing the defect. A cell hybrid generated by the fusion of CHO cells and L cell fibroblasts was used for comparison due to their competency in processing Ag. The transport pathway of cysteine within the CHO cells appeared normal. However, these cells had a significantly lower level of glutathione, a major physiological reducing thiol, compared to the cell hybrid. Treatment of the CHO cells with N-acetyl-L-cysteine did not augment their glutathione content nor their ability to process Ag. When the cell hybrid was treated with L-buthionine-(S,R)-sulfoximine (BSO), which significantly decreased their glutathione level, the hybrid poorly processed hen egg lysozyme (HEL) and ovalbumin, which have disulfide bonds. In contrast, BSO treatment did not affect the capacity of the hybrid to process pigeon cytochrome c and carboxymethylated HEL, which lack disulfide bonds. Therefore, low intracellular glutathione levels in antigen-presenting cells correlated with defective processing of Ag with disulfide bonds, indicating that this thiol may be a critical factor in regulating productive Ag processing.

Acetylcysteine↗
PubMed1996

Gallium arsenide augments antigen processing by peritoneal macrophages for CD4+ helper T cell stimulation.

Gallium arsenide (GaAs) suppresses numerous immunologic functions of leukocytes at distal locations from the exposure site. The effect of direct GaAs exposure on peritoneal cells was examined by i.p. administration of the chemical. No alteration in the frequency of various cell lineages, including macrophages, B cells, CD4+ and CD8+ T cells, and granulocytes, occurred within the peritoneal population from GaAs-exposed mice. The ability of macrophages to function as antigen-presenting cells (APC) by processing a panel of soluble protein antigens was investigated by the stimulation of antigen-specific helper T cell hybridomas to secrete interleukin-2. On a per cell basis, GaAs-exposed macrophages were more efficient than vehicle control cells in activating the T cells with all native antigens examined. GaAs exposure increased the expression of major histocompatibility complex class II molecules, which are recognized by these T cells, on the surface of macrophages. However, the level of T cell activation with peptide fragments of the antigens, which do not require processing, was not enhanced with GaAs-exposed cells as APC. In contrast, the capability of latex bead-exposed macrophages to function as APC was comparable to that of vehicle control cells, suggesting that GaAs modulatory effects were not merely due to phagocytosis of particles. These findings suggest that direct GaAs exposure augments antigen processing, but not presentation, by macrophages perhaps by activating the cells, which may contribute to the immunotoxicity and inflammatory reaction caused by respiratory exposure to GaAs.

Animals↗
PubMed1996

Augmented antigen presentation by mouse Ia + T clone cells BK-BI-2.6.O4.1 mediated by transferrin receptors.

The murine T clone cells BK-BI-2.6.O4.1 (BI/O4.1) synthesize and express MHC class II molecules constitutively. BI/O4.1 cells are able to present various protein antigens to antigen-specific CD4 + T cells. However, a 10-fold higher concentration of antigen is needed to activate specific T cells to lymphokine secretion by BI/O4.1 cells in comparison with spleen cells or with the more homogeneous population of bone marrow-derived macrophages (BMMph). The authors tested whether the reduced antigen presentation potential of BI/O4.1 cells was augmented by transferrin-mediated uptake of the model antigen ovalbumin (OVA) coupled to human ferric transferrin. It was shown that 240-fold less OVA was sufficient to induce proliferation of an OVA-specific T-cell clone when the conjugate and not native OVA was used. The presence of ferric TF in the cultures competitively inhibited this effect of the conjugate. A similar shift in the dose-response curve to lower doses of antigen was induced by the conjugate when B lymphoma cells were used as antigen-presenting cells. BMMph and P388D1 cells processed and presented the conjugate with similar efficiency as native OVA, although both cell types exposed transferrin receptors. These data suggest that the reduced antigen presentation potential of BI/O4.1 T clone cells is due to the inefficient uptake of OVA by pinocytosis and delivery into the processing compartment.

Animals↗
PubMed1996

delta 9-Tetrahydrocannabinol selectively inhibits macrophage costimulatory activity and down-regulates heat-stable antigen expression.

delta 9-Tetrahydrocannabinol (THC) exposure inhibits numerous immunologic functions of macrophages. The ability of THC-exposed macrophages to provide costimulatory signals to helper T cell hybridomas was investigated by induction of interleukin-2 secretion by T cells in response to immobilized monoclonal anti-CD3 antibody. Exogenous interleukin-1 did not deliver a costimulatory signal to these T cells, suggesting that macrophage costimulatory activity was mediated through cell surface molecules. Modulation of the T cell responses by THC depended on the source of costimulation. THC did not suppress costimulatory activity provided by peritoneal macrophages or immobilized fibronectin. THC at low concentrations markedly diminished the costimulatory activity of a macrophage hybridoma to activate one T cell but not another. Inhibition of costimulation by THC inversely correlated with the loss of activity caused by paraformaldehyde fixation of macrophages. THC at 10(-8) M significantly decreased expression of costimulatory heat-stable antigen, which is resistant to fixation, on the macrophage hybridoma. However, expression of costimulatory B7-1 and B7-2 molecules, which are sensitive to fixation, was not affected by THC. Therefore, THC selectively suppresses a fixation-resistant costimulatory signal to helper T cells in part by diminishing expression of heat-stable antigen.

Animals↗
PubMed1996

Gallium arsenide selectively suppresses antigen processing by splenic macrophages for CD4+ T cell activation.

Gallium arsenide (GaAs) is an intermetallic compound used in the electronics industry as a semiconductor. Acute exposure of animals to GaAs suppresses various immune functions. We investigated the effects of GaAs on immunocompetency with emphasis on macrophages. Mice were given 12.5 to 200 mg/kg GaAs i.p., and immune parameters were examined 1 or 5 days later. Chemically exposed mice did not display alteration in spienic cellular composition. Despite this, primary in vitro humoral response to sheep red blood cells by GaAs-exposed mice was inhibited in a dose-dependent manner. The ability of 5-day vehicle- or 200 mg/kg GaAs-exposed splenic macrophages to induce interleukin-2 production by antigen-specific CD4+ helper T cell hybridomas stimulated with soluble protein antigens was assessed. GaAs-exposed macrophages were less competent in eliciting T cell responses to pigeon cytochrome c and pork insulin than vehicle-exposed cells. However, GaAs-exposed macrophages activated hen egg lysozyme- and chicken ovalbumin-specific T cells as efficiently as vehicle control cells. Also, suppressed processing of cytochrome c was not observed after a 1-day exposure. Chemical exposure did not alter the expression of major histocompatibility complex class II molecules on the macrophages or their activation of T cells by peptides, which do not require processing. Therefore, GaAs causes a time- and antigen-dependent defect in antigen processing that is essential for CD4+ T cell stimulation by splenic macrophages.

Animals↗
PubMed1995

Characterization of fibroblasts with a unique defect in processing antigens with disulfide bonds.

A chinese hamster ovary (CHO) fibroblast, transfected with murine MHC class II genes, inefficiently stimulated CD4+ Th cells specific for OVA, hen egg lysozyme (HEL), and pork insulin which contain disulfide bonds. However, the fibroblasts elicited a T cell response to lambda repressor, which lacks disulfide bonds, and efficiently presented synthetic peptides. A somatic cell hybrid WALC, generated by fusing the hamster fibroblast with a murine L cell fibroblast, very efficiently processed OVA and HEL, suggesting that impaired processing was genetically complemented and was a recessive trait. The hamster fibroblasts were capable of processing two distinct denatured forms of OVA and carboxymethylated HEL, either as effectively or more efficiently than the B lymphoma cell. The CHO cells also displayed diminished disulfide reduction of an endocytosed [125I]tyramine linked to poly-(D-lysine) through a disulfide spacer compared with that of the cell hybrid, providing direct evidence for defective reductive cleavage by the CHO cells. Diminished aspartic acid-mediated proteolysis of Ag could not account for the phenotype, because cell lysates and separated organelles from the fibroblast possessed higher acidic aspartyl proteolytic activity than lysates and organelles from a B lymphoma cell. Thus, CHO cells exhibit a defect in processing Ag with disulfide bonds which is consistent with the impaired intracellular reduction of the disulfide bonds in endocytosed macromolecules.

Animals↗
PubMed1995

delta 9-Tetrahydrocannabinol modulates antigen processing by macrophages.

delta 9-Tetrahydrocannabinol (THC) exposure suppresses multiple immunological functions of macrophages. The ability of macrophages exposed to THC to process and present soluble protein antigens was investigated by the stimulation of antigen-specific helper T cell hybridomas to secrete interleukin-2. The T cell response to hen egg lysozyme was dramatically reduced after a 24-hr pretreatment of a macrophage hybridoma with THC. In contrast, THC exposure did not alter the capacity of the macrophage hybridoma to process chicken ovalbumin and augmented their presenting cell function for a pigeon cytochrome c response. These findings could not be attributed to differential effects of THC on either cell viability or expression of the antigen receptor-associated CD3 complex by the T cells. The level of T cell activation with peptides of lysozyme and cytochrome c, which do not require processing, was inhibited only at the highest concentrations of THC, suggesting that THC mainly affects antigen processing. Peritoneal macrophages exposed to THC during an antigen pulse and fixed with paraformaldehyde showed similar effects on the subsequent T cell responses to lysozyme and cytochrome c in the absence of THC, arguing against a possible influence of THC on the T cells. Therefore, THC differentially modulates the capacity of macrophages to process antigens that is necessary for the activation of CD4+ T cells.

Animals↗
PubMed1993

Differences among various lineages of antigen-presenting cells in processing exogenous antigen internalized through transferrin receptors.

The Ag, pigeon cytochrome c, was coupled to human ferric transferrin by a heteroligation technique to target Ag into the endosomal transport pathway via transferrin receptors. The ability of various types of APC that do or do not express transferrin receptors to process exogenous Ag in their endosomes was investigated by the stimulation of Ag-specific CD4+ T cells with the transferrin-Ag conjugate in a serum-free assay. When two B lymphoma cells were the source of APC, the conjugate was significantly more potent than native Ag in activating the T cells, agreeing with our previous finding using a third B lymphoma cell. The conjugate and Ag were similarly presented by splenic B cells that lack transferrin receptors to the T cells. However, both a macrophage hybridoma and a MHC class II-L cell transfectant hardly elicited a T cell response to the conjugate, although a response to native Ag was readily observed. These findings could not be attributed to an absence of transferrin receptors or receptor-mediated internalization of the conjugate, nor to differential expression of MHC class II molecules or li chain by the APC. The poor presentation of the conjugate by the L cell transfectants was associated with diminished catabolism of the conjugate, however, the macrophage hybridoma rapidly degraded the conjugate, similar to the B lymphoma cell. Peritoneal macrophages, which lack transferrin receptors, and the macrophage hybridoma induced a response to the conjugate only at concentrations that allowed internalization by fluid phase pinocytosis. The lower potency of the conjugate compared with native Ag with non-B-presenting cells suggest that these cell types process the conjugate by a different mechanism than used by B cells. Differences in the mechanism of Ag processing used by APC of distinct cell lineages may possibly influence immune responsiveness.

Animals↗