Biomedical subjects

S P Colgan

Publications and source records attributed to S P Colgan.

65 records · Page 4Linked to original sources

PubMed1993

5'-adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers.

Neutrophil transmigration across intestinal epithelia is thought to contribute to epithelial dysfunction and characterizes many inflammatory intestinal diseases. Neutrophils activated by factors, normally present in the lumen, release a neutrophil-derived secretagogue activity to which intestinal epithelia respond with an electrogenic chloride secretion, the transport event which underlies secretory diarrhea. Using sequential ultrafiltration, column chromatographic, and mass and Raman spectroscopic techniques, neutrophil-derived secretagogue was identified as 5'-AMP. Additional studies suggested that neutrophil-derived 5'-AMP is subsequently converted to adenosine at the epithelial cell surface by ecto-5'-nucleotidase and that adenosine subsequently activates intestinal secretion through adenosine receptors on the apical membrane of target intestinal epithelial cells. These findings suggest that this ATP metabolite may serve as a neutrophil-derived paracrine mediator that contributes to secretory diarrhea in states of intestinal inflammation.

Adenine Nucleotides↗

PubMed1993

Lipoxin A4 modulates transmigration of human neutrophils across intestinal epithelial monolayers.

Neutrophil (PMN) migration across intestinal epithelial barriers, such as occurs in many disease states, results in modifications in epithelial barrier. Here, we investigated the impact of lipoxin A4 (LXA4), an eicosanoid with counterregulatory inflammatory roles, on PMN migration across cultured monolayers of the human intestinal epithelial cell line T84. Transepithelial migration of PMN was assessed in the apical-to-basolateral direction and in the basolateral-to-apical direction. In the apical-to-basolateral direction, preexposure of PMN to LXA4 (10 nM, 15 min) stimulated an 87 +/- 5% increase in transepithelial migration of PMN as determined by a PMN myeloperoxidase assay. The LXA4-elicited effect on transmigration was present throughout the 2-h assay period and was not secondary to LXA4 effects on epithelial monolayer integrity as judged by measurement of transepithelial resistance. PMN migration in the basolateral-to-apical direction was modulated by LXA4 with a comparable time- and concentration-dependence to that in the apical-to-basolateral direction. However, qualitative differences in how LXA4 modulates transmigration in the two opposing directions were observed. In the basolateral-to-apical direction, preexposure of PMN to LXA4 (10 nM, 15 min) diminished PMN transepithelial migration by 33 +/- 4%. Structure-function studies revealed that LXA4 and 11-trans-LXA4 (50% of LXA4 effect), but not LXB4, inhibited basolateral-to-apical PMN transmigration. The action of LXA4 was not sensitive to inhibitors of cyclooxygenase or specific leukotriene biosynthesis, but was sensitive to staurosporine, a protein kinase C inhibitor. These results suggest that migration of PMN across epithelia in the physiological direction may be qualitatively different following PMN exposure to eicosanoids. We propose that such retention of PMN at this specific anatomic location may serve an important role in mucosal defense.

Cell Line↗

PubMed1992

The myeloid leukemia cell line HL-60 produces a factor that induces chloride secretion in cultured epithelial cells.

When activated, polymorphonuclear leukocytes (PMNs) produce a small soluble factor, termed neutrophil-derived secretagogue (NDS), that elicits electrogenic Cl- secretion--the transport event responsible for hydration of mucosal surfaces. Work toward purification of this factor has been hampered by variability in activity of PMN-derived NDS. Using a human-derived intestinal epithelial cell line (T84) that serves as a model for studies of the regulation of electrogenic Cl- secretion, we find that the human promyelocytic leukemia cell line HL-60 secrets a factor with NDS-like activity. Buffer conditioned by HL-60 cells (10(7) cells/ml for 1 h), when applied to T84 monolayers grown on permeable supports and analyzed by routine electrophysiological techniques, elicited a short-circuit current (Isc) of 11.7 +/- 1.02 microA/cm2. This short-circuit current was sensitive to bumetanide, an inhibitor of the basolateral Na-K-2Cl cotransporter, and was dependent on the presence of chloride in the assay buffers. Such data indicate that buffer conditioned by HL-60 cells stimulates electrogenic Cl- secretion. Like NDS, the active factor in HL-60-conditioned buffer has a nominal molecular weight of less than 500 and was increased by activation of cells with phorbol ester. 125I and 86Rb efflux assays confirmed that the secretagogue released by stimulated HL-60 cells, similar to PMN-derived NDS, preferentially stimulates opening of Cl- rather than K+ channels in T84 cells. Lastly, release of NDS-like bioactivity increases when HL-60 cells are differentiated toward granulocytes compared to cells differentiated toward monocytes.

Cells, Cultured↗

PubMed1992

Defective in vitro motility of polymorphonuclear leukocytes of homozygote and heterozygote Chediak-Higashi cats.

The in vitro migratory responses of neutrophils of homozygote and heterozygote Chediak-Higashi cats were defective in an under-agarose assay when compared to the behavior of phagocytes of control cats. The linear distances traversed by the leading front of migrating Chediak-Higashi neutrophils toward streptococcal culture supernatant, zymosan-activated serum or buffer were reduced and smaller numbers of Chediak-Higashi phagocytes populated the resulting migration areas than did cells of control animals. The relative migration parameters of the Chediak-Higashi phagocytes, however, did not differ from the corresponding parameters of control neutrophils in the presence of streptococcal culture supernatant. Therefore, phagocytes of homozygote and heterozygote Chediak-Higashi cats recognized and responded equally well to the bacterial stimuli as did cells of control animals but traveled shorter distances primarily because of a reduced inherent motility. Similar results were also obtained when the feline phagocytes were attracted by zymosan-activated serum. In addition the relative migration parameters of the neutrophils of homozygote Chediak-Higashi cats were reduced and the normalized spatial distributions of their migrating cells were significantly different in the presence of 100% and 20% zymosan-activated serum when compared to the corresponding migration parameters of carrier and control animals. Defective recognition or responses to the higher concentrations of these host-derived attractants complicated, therefore, the already reduced inherent motility of the phagocytes of homozygote Chediak-Higashi cats.

Animals↗

PubMed1992

Neutrophil migration across a cultured epithelial monolayer elicits a biphasic resistance response representing sequential effects on transcellular and paracellular pathways.

Migration of polymorphonuclear leukocytes across epithelia is a hallmark of many inflammatory disease states. Neutrophils traverse epithelia by migrating through the paracellular space and crossing intercellular tight junctions. We have previously shown (Nash, S., J. Stafford, and J.L. Madara. 1987. J. Clin. Invest. 80:1104-1113), that leukocyte migration across T84 monolayers, a model human intestinal epithelium, results in enhanced tight junction permeability--an effect quantitated by the use of a simple, standard electrical assay of transepithelial resistance. Here we show that detailed time course studies of the transmigration-elicited decline in resistance has two components, one of which is unrelated to junctional permeability. The initial decrease in resistance, maximal 5-13 min after initiation of transmigration, occurs despite inhibition of transmigration by an antibody to the common beta subunit of neutrophil beta 2 integrins, and is paralleled by an increase in transepithelial short-circuit current. Chloride ion substitution and inhibitor studies indicate that the early-phase resistance decline is not attributable to an increase in tight junction permeability but is due to decreased resistance across epithelial cells resulting from chloride secretion. Since T84 cells are accepted models for studies of the regulation of Cl- and water secretion, our results suggest that neutrophil transmigration across mucosal surfaces (for example, respiratory and intestinal tracts) may initially activate flushing of the surface by salt and water. Equally important, these studies, by providing a concrete example of sequential transcellular and paracellular effects on transepithelial resistance, highlight the fact that this widely used assay cannot simply be viewed as a direct functional probe of tight junction permeability.

Cell Movement↗

PubMed1992

Neutrophil function in normal and Chediak-Higashi syndrome cats following administration of recombinant canine granulocyte colony-stimulating factor.

The effects of recombinant canine granulocyte colony-stimulating factor (rcG-CSF) on leukocyte counts and neutrophil function in clinically normal cats and in cats heterozygotic and homozygotic for Chediak-Higashi syndrome (CHS) were examined. CHS is a genetic disease characterized by neutropenic episodes and defects in a variety of phagocyte functions. Short-term administration of rcG-CSF at 10 micrograms/kg body weight resulted in a five- to tenfold increase in circulating granulocytes by day 10 of administration and normalizes CHS neutrophil counts by day 3. The drug was specific for neutrophils as determined by differential cell counts. Neutrophil chemotaxis under agarose and phagocytosis of Escherichia coli were characterized following administration of rcG-CSF in vivo. Granulocytes elicited by rcG-CSF show enhanced chemotactic abilities toward activated serum, increased spontaneous migration, and an enhanced ability to ingest opsonized E. coli. At a concentration of 1 nM rcG-CSF in vitro, chemotaxis and spontaneous migration were increased, with no effect on phagocytosis. CHS neutrophil function was improved by administration of rcG-CSF in all parameters studied, although the defect in chemotaxis was present throughout the treatment period. We conclude from this study that neutrophils elicited by rcG-CSF are functionally enhanced and that rcG-CSF may be a viable therapy for CHS and other related disorders.

Animals↗

PubMed1992

Effects of low-dose aspirin and specific thromboxane synthetase inhibition on whole blood platelet aggregation and adenosine triphosphate secretion in healthy dogs.

Platelet aggregation and adenosine triphosphate (ATP) secretion in response to arachidonic acid (10 microM) or collagen (5 micrograms/ml) were compared in healthy, adult female Beagles treated with low-dosage aspirin (3.5 mg/kg of body weight, PO, q 12 h for 7 treatments) or with CGS 12970, a specific thromboxane synthetase inhibitor (10 mg/kg, PO, q 8 h for 10 treatments). Platelet aggregation was assessed in whole blood by use of an electrical impedance method. Baseline values obtained prior to treatment served as controls. Addition of arachidonic acid to blood from nontreated dogs resulted in significantly (P less than 0.001) increased impedance, but had no effect in blood from dogs treated with either aspirin or CGS 12970. Treatment with CGS 12970 or aspirin significantly (P less than 0.001) decreased platelet ATP secretion in response to arachidonic acid, compared with baseline values; however ATP secretion in aspirin-treated dogs was significantly (P less than 0.01) less than ATP secretion in CGS 12970-treated dogs. Differences in platelet aggregation were not observed between control dogs and aspirin- or CGS 12970-treated dogs in response to collagen as an aggregant, however, collagen-induced platelet ATP secretion was significantly (P less than 0.001) decreased in dogs treated with aspirin, compared with control values and values from dogs treated with CGS 12970. In dogs treated orally with 0.1, 0.2, 1.0, or 10 mg of CGS 12970/kg, dose-dependent inhibition of arachidonic acid-induced platelet aggregation was observed, with impedance changes not observed at the 10-mg/kg dosage and normal platelet aggregation associated with the 0.1-mg/kg dosage.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1991

Restoration of neutrophil and platelet function in feline Chediak-Higashi syndrome by bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) was successfully performed in four Chediak-Higashi (CHS) syndrome affected cats. Preparatory regimens included selective intestinal flora decontamination, fractionated total body irradiation for myeloablation, and prophylactic treatment for graft-versus-host disease with cyclosporin A. Neutrophil chemotaxis under-agarose and whole-blood platelet aggregation/secretion were characterized prior to BMT and after engraftment of donor-origin marrow cells. Liver and kidney biopsies were obtained and evaluated by light and electron microscopy before, and at 6 months post-BMT to determine what effect BMT might have on abnormal lysosome fusion in hepatocytes and renal tubule cells. The platelet storage pool defect was resolved by day 40 post-BMT. In vitro neutrophil migration in all cats appeared to improve with time after BMT and complete restoration was evident by day 175 post-BMT. No apparent differences were evident in either the liver or the kidney at 6 months post-BMT. One cat developed seizures and one developed posterior paresis 5 months post-BMT; neurologic impairment ultimately resulted in death of two cats at 6 and 8 months post-BMT, respectively. Neurologic lesions in both cats were characterized by non-suppurative encephalitis. Allogeneic BMT successfully corrected the neutrophil migration defect and platelet storage pool deficiency but had no effect on lysosome distribution in liver and kidney cells of CHS cats.

Adenosine Triphosphate↗

PubMed1989

Influence of technical parameters on the in vitro motility of equine neutrophils in the presence of streptococcal culture supernatant.

To identify the influence of technical factors on the in vitro motility of equine neutrophils towards streptococcus culture supernatant in an under-agarose assay, we studied the changes in eight cell migration parameters. The distances the phagocytes travelled by directed, random and spontaneous migration increased with incubation time, cell concentration and the gelatin and serum contents of the migration plates. The contribution of chemotaxis to the phagocyte migrations, however, decreased simultaneously. The directed and random, though not the spontaneous, migrations of the phagocytes increased also when the chemoattractant wells were placed closer to the cell wells but so did the influence of the chemokinetic activity of the bacterial culture supernatant on phagocyte motility. In contrast, preincubation of migration plates with the chemoattractant, the agarose content of the migration plates and contamination of the granulocytes with non-migrating, mononuclear cells did not substantially affect the in vitro migrations of the neutrophils. The changes in the in vitro motility of the equine neutrophils by these technical factors were, in general, comparable to those reported for human cells attracted by a variety of host-and bacteria-derived chemoattractants.

Animals↗

PubMed1989

Platelet aggregation and ATP secretion in whole blood of normal cats and cats homozygous and heterozygous for Chediak-Higashi syndrome.

A rapid and simple technique using the Whole Blood Lumi-Aggregometer was used to study storage pool disease in Chediak-Higashi homozygote and heterozygote cats. Feline Chedlak-Higashi platelets aggregated after the addition of both ADP and collagen. During platelet aggregation, ATP secretion was assayed; the whole blood aggregometer is effective in detecting decreased levels of secretable ATP in homozygote cats. No storage pool deficiency was found in heterozygote cats. However, upon analysis of impedance tracings, a decreased platelet aggregation response was seen in both homozygote and heterozygote cats. These results suggest that prolonged bleeding times in Chediak-Higashi cats may involve a mechanism in addition to a dense granule deficiency.

Adenosine Triphosphate↗

PubMed1988

Chemotactic and chemokinetic activity of Streptococcus faecalis culture supernatant for equine neutrophils.

Although equine neutrophils did not respond towards formylated methionyl peptides, Streptococcus faecalis culture supernatant caused an in vitro stimulation of equine neutrophil motility when measured by an under-agarose assay. The migration of neutrophils towards the culture supernatant increased sigmoidally with the logarithmic concentration of the culture supernatant in the chemoattractant wells. The streptococcal culture supernatant was chemokinetic because it stimulated the random motility of the phagocytes. Because granulocytes migrated further towards the supernatant than could be explained by the chemokinetic activity of the bacterial products, the streptococcal culture fluid also exerted a chemotactic effect on the leukocytes. The chemotactic activity of the supernatant was further confirmed by the changes in the orientation of the migrating cells during incubation. These results indicate that bacteria produce cytotaxins other than formylmethionyl peptides which are recognized by equine neutrophils.

Animals↗