Biomedical subjects

A Skeel

Publications and source records attributed to A Skeel.

35 records · Page 2Linked to original sources

PubMed1993

Secretion of monocyte chemoattractant protein-1 (MCP-1) by human mononuclear phagocytes.

Concentrations of MCP-1 and NAP-1 in culture fluids of human leukocytes were measured by sandwich ELISA. PPD caused PBMC's from tuberculin-sensitive subjects to secrete MCP-1 and NAP-1. PPD did not stimulate secretion by cells from a tuberculin-negative subject. Since the amounts secreted were more than could be produced by the few PPD-sensitized lymphocytes in the culture, we postulate that other cells were stimulated to secrete these chemoattractants. This study evaluated secretory capacity of one of the cell types in the PBMC culture. Unstimulated monocytes did not secrete MCP-1 or NAP-1. In order of increasing effect, IL-2 + IFN gamma, IL-1 alpha, and LPS caused monocyte secretion of MCP-1. The rank order for NAP-1 secretion was the same. TNF alpha did not cause secretion of MCP-1, but caused about the same amount of NAP-1 secretion as IL-2 + IFN gamma. Composition of the culture medium was especially critical for LPS-induced secretion of MCP-1, which was greatly enhanced by FCS and by Iscove's DMEM compared to RPMI 1640. IL-4 inhibited LPS-induced secretion of both MCP-1 and NAP-1. Secretory patterns were also a function of mononuclear phagocyte phenotype. LPS-induced secretion of MCP-1 was much greater for monocytes cultured several days in CSF-1 than for freshly isolated monocytes. LPS stimulation of bronchoalveolar macrophages caused NAP-1 secretion, but no secretion of MCP-1 above a relatively low baseline level.

Chemokine CCL2↗

PubMed1991

Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity.

Macrophage stimulating protein (MSP) was purified to homogeneity from human blood plasma by selection of biologically active fractions obtained by sequential immunoaffinity and high pressure liquid ion exchange chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular mass of MSP was 70 kilodaltons (kD); under reducing conditions two gel bands were seen, at 47 and 22 kD. The disulfide-linked two-chain structure of MSP was confirmed by separation of reduced and alkylated MSP chains. A computer search comparison of six partial sequences of MSP digests showed that MSP has not been recorded in data banks of protein sequences. Two MSP fragments had greater than 80% identity in overlaps of 12-16 residues to sequences in the protein family that includes human prothrombin, plasminogen, and hepatocyte growth factor. The concentration of purified MSP required for half-maximal biological activity was the order of 10(-10) M. In addition to making mouse resident peritoneal macrophages responses to chemoattractants, MSP caused the appearance of long cytoplasmic processes and pinocytic vesicles in freshly plated macrophages. MSP also caused phagocytosis via the C3b receptor, CR1. Whereas resident peritoneal macrophages bind but do not ingest sheep erythrocytes opsonized with IgM anti-Forssman antibody and mouse C3b, addition of MSP caused ingestion. Thus, MSP causes direct or indirect activation of two receptors of the mouse resident peritoneal macrophage, CR1 and the C5a receptor.

Amino Acid Sequence↗

PubMed1991

Biological aspects of monocyte chemoattractant protein-1 (MCP-1).

In this communication, we have asked if MCP-1 is the mediator of cellular infiltration in DCH, outlining the criteria in Table 3. Preliminary data suggest that PHA-stimulated lymphocytes secrete MCP-1, and that MCP-1 can be produced in response to antigen stimulation. MCP-1 attracts monocytes and basophils, but not neutrophils. The question of a lymphocyte response to MCP-1 requires further study. We have emphasized that the discovery of leukocyte-specific NAP-1 and MCP-1 should now be followed by exploration of conditions in which one agonist is secreted without the other. This would be expected, for example, in DCH, which is characterized by mononuclear leukocyte infiltration without neutrophils.

Basophils↗

PubMed1990

Leukocyte specificity and binding of human neutrophil attractant/activation protein-1.

Neutrophil attractant/activation protein-1 (NAP-1) was previously shown to attract human neutrophils, but not monocytes. The purpose of this study was to determine if NAP-1 interacted with other types of blood leukocytes. In addition to its chemotactic activity for neutrophils, NAP-1 induced chemotactic responses by T lymphocytes and basophils. Chemotactic potency (10(-8) M for an optimal response) was the same for all three cell types. However, NAP-1 caused a chemotactic response in excess of random migration of 7% or 16% of basophils (depending on the medium used) and only 9% of T lymphocytes, in contrast to 30% of neutrophils. This agonist was not chemotactic for partially purified normal human eosinophils. The symmetrical histogram obtained by flow cytometry of neutrophils equilibrated at 0 degree C with fluoresceinated NAP-1 indicates that all neutrophils bound the ligand. A dose-response curve plateau, and inhibition of binding of NAP-1-FITC by unlabeled ligand are evidence for saturable binding to receptors, estimated to be 7000 per cell. Our results suggest that, for induction of an acute inflammatory response, the quantitatively significant action of NAP-1 is on neutrophils.

Basophils↗

PubMed1990

A multiwell cell settling and adherence chamber for morphology and differential counting.

A simple multiwell chamber is described that can be used to prepare randomly distributed cells on a microscope slide, suitable for morphological identification and differential counting. To the eight wells of the chamber are added 50-microliter volumes of cell suspension at concentrations of 10(3)-10(6) cells/ml. As the cells settle, fluid is slowly wicked away by a damp filter paper sandwiched between the microscope slide and the acrylic top plate of the multiwell chamber. Within 20-40 minutes, the cell monolayers on the slide are completely dry. The combined settling and bulk fluid removal results in a distribution of adherent cells that are sufficiently spread to exhibit excellent morphology after staining. If the chamber is centrifuged for 30 seconds at 50x g immediately after addition of cells, recovery of cells in the monolayer is virtually 100%, and as few as 50 input cells per 50 microliters can be detected. Agreement between predicted and observed differential counts of cell mixtures indicates that cells in the monolayer were distributed randomly.

Cell Adhesion↗

PubMed1989

Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different lengths of the amino-terminal sequence.

Human monocyte-derived neutrophil chemotactic factor (MDNCF) was purified from culture supernatant of lipopolysaccharide-stimulated human peripheral blood mononuclear leukocytes on a column of Sepharose-bound murine monoclonal anti-MDNCF. About 65% of the culture fluid chemotactic activity was bound to the column. The unbound 35% probably represents chemotactic activity of other cytokines in the culture fluid. More than 85% of the bound activity was eluted by pH 2.5 glycine buffer. When this material was applied to an HPLC-CM column, gradient elution produced four well-separated A280 peaks, each of which had chemotactic activity. N-terminal amino acid analysis of the four peaks revealed three different sequences. One (MDNCF-c) was identical to the sequence that we reported previously. The other two (MDNCF-a and -b) had seven and five additional amino acids, respectively, at the N-terminus. MDNCF-a, -b and -c accounted for 8, 47 and 45% of the total MDNCF peptide. Alignment with the MDNCF cDNA sequence shows that MDNCF-a results from cleavage of a 20 residue signal peptide. MDNCF-c results from culture fluid proteolytic cleavage of the N-terminal sequences of MDNCF-a and -b at an R-S bond. The three peptides occurred in the four HPLC-CM peaks in different ratios. The bulk of any one peptide was distributed in two adjacent HPLC-CM peaks. This suggests that each peptide exists in a minimum of two states. In contrast to our previous multi-step purification, the immunoaffinity and HPLC-CM column sequence resulted in complete purification of MDNCF in two steps and led to identification of two additional MDNCF peptides, one of which has not heretofore been detected.

Amino Acid Sequence↗

PubMed1987

Dynamics of chemotactic peptide-induced superoxide generation by human monocytes.

Addition of the chemotactic peptide, f-Met-Leu-Phe, to human monocytes induced a burst of superoxide release, which ceased after approximately 3 min. Diminished responsiveness to f-Met-Leu-Phe, but not to phorbol myristate acetate (PMA), was induced by 1- to 3-h storage at 0 degrees C or by 2 min in 40 microM adenosine (ADO). Reversal of the ADO block was achieved by addition of adenosine deaminase (ADA) as little as 15 sec before the f-Met-Leu-Phe stimulus; ADA had no effect when added poststimulus. The ADO experiments suggest that there are a minimum of two sequentially produced intermediates in the f-Met-Leu-Phe stimulus-response pathway. The first intermediate persists for less than 30 sec. The second, formation of which is stimulated by the first, persists for the duration of the response and is the target of ADO inhibition. The ADO target is apparently not protein kinase-C, since the response of inhibited cells to PMA was unimpaired. The maximal inhibition by adenosine of f-Met-Leu-Phe-induced superoxide generation was approximately 50%. It is possible that f-Met-Leu-Phe stimulates two pathways of NADPH activation, only one of which is inhibited by adenosine.

Adenosine↗

PubMed1985

Disposable microliter immunoabsorbent columns: construction and operation.

Disposable microliter immunoabsorbent columns were constructed from pasteur pipets. The bed support was a cube of gelatin surgical sponge, which was tamped into the pipet tip. Column dead space, represented by the compressed volume of the sponge, was 5 microliters. The columns were used with protein A-Sepharose; settled bed volumes were 50 microliters. It was possible to pour columns that functioned as immunoabsorbents with bed volumes as small as 10 microliters. There was no gravity flow through these columns. Flow was achieved by touching column tips to absorbent paper if liquid was to be discarded or to 50 microliters capillary tubes for fluid collection. A simple capillary collection tube assembly was designed for operation of a row of 10 columns at a time. In a test system of [3H]methotrexate and IgG anti-methotrexate, 90% of applied antigen was bound to antibody columns, whereas 90% was recovered in the eluates from control columns. The columns were used in the initial step of screening uncloned hybridoma culture fluids for anti-MSP.

Animals↗

PubMed1985

Analysis of human monocyte chemoattractant binding by flow cytometry.

The chemotactic peptide fMet-Leu-Phe-Lys is a potent chemoattractant for human blood monocytes. However, only one-third of the monocytes respond. To determine whether or not lack of response reflected absence of attractant receptors, we equilibrated peripheral blood mononuclear cells with fMet-Leu-Phe-Lys-FITC and analyzed binding by flow cytometry. The fluoresceinated peptide bound rapidly at 0 degree C, and the amount bound approached saturation with increasing concentration. The percentage of blood monocytes that bound the peptide was 60 +/- 8 (SEM for seven experiments). In contrast, only 36 +/- 3% (SEM for 16 experiments) of monocytes responded to the attractant by directed migration. It follows that, among the 64 nonmigrating monocytes per 100 total monocytes, approximately 40, or two-thirds of them, fail to bind attractant; the remaining one-third bind attractant but do not respond with directed movement.

Chemotaxis, Leukocyte↗

PubMed1985

Separation of human basophils into two fractions with different density and histamine content.

We designed experiments in this study to test the hypothesis suggested by recent purification data that blood basophils comprise two populations of different density, which circulate in numbers characteristic for each human subject. Basophils were separated into two density bands by single step centrifugation on a discontinuous Percoll gradient. Band 1 cells were at the interface between plasma and Percoll of density 1.070 gm/ml. Band 2 cells were at the Percoll 1.070 to 1.080 interface. When the number of band 1 basophils was expressed as a percentage of the total in bands 1 and 2, this relative amount generally remained in a narrow range for blood obtained from the same donor on 3 successive days but differed markedly in different individuals. In a series of leukapheresis experiments, we demonstrated that the percentage of band 1 basophils in postleukapheresis venous blood was strikingly similar to the preleukapheresis value. If basophils that repopulated the leukapheresis-depleted circulation came from the bone marrow, we can conclude that blood levels of basophils in bands 1 and 2 are under physiologic control and that the two types of basophils are released in amounts characteristic for each human subject. Additional evidence for two distinct blood basophil populations was provided by histamine measurements. The histamine content per basophil was consistently higher in cells from band 1 than from band 2, the mean difference between pairs of values for 30 subjects being 0.3 +/- 0.04 pg or about 27% of the band 1 basophil histamine content of 1.1 pg.

Basophils↗

PubMed1984

Purification of human blood basophils by single step isopycnic banding on Percoll.

Human venous blood, anticoagulated with EDTA, was layered onto a discontinuous Percoll gradient, made from solutions of density 1.088, 1.079, and 1.070 gm/ml. After centrifugation at 700g for 15 min at 22 degrees C, the majority of the blood basophils was found in a narrow band at the density 1.070-1.079 interface (Percoll band 2). For 15 normal donors, mean total basophil number recovered from all locations in the gradient was 3.8 +/- 1.2 (SD) X 10(4) basophils per ml of blood applied. Thus, 95% of the values ranged from 1.5 to 6 X 10(4), which compares favorably with the reported range of 1 to 8 X 10(4) basophils per ml for normal subjects. In the basophil-rich Percoll band 2, 2.8 +/- 0.8 X 10(4) basophils were recovered per ml of blood applied. The mean percentage of basophils in Percoll band 2 was 19%, with a range of 5 to 53%. Monocytes and neutrophils were present in very small numbers; the majority of accompanying cells were small lymphocytes.

Basophils↗

PubMed1983

A high molecular weight chemoattractant generated from C5 by ultracentrifugation of mouse serum without activation of complement.

After ultracentrifugation of normal mouse serum, we found chemoattractant activity in the high molecular weight protein region at the bottom of the tube, which was comparable in amount and potency to the attractant in endotoxin-activated serum. This was not a pre-formed attractant, but was generated from serum reactants at least one of which was inactivated by heating at 56 degrees C. Analysis of sera from 10 different mouse strains for hemolytic C5 activity and for capacity to generate chemoattractant on ultracentrifugation showed that the 4 strains without C5 were the only strains that failed to generate the attractant. Thus, the attractant precursor is C5. Since the activity was generated in the presence of 0.01 M EDTA, classical or alternative complement activation was not required. The chemoattractant product had a mol. wt of approximately 170,000; it was therefore not free C5a. These results, and data recently published on digestion of purified human C5 by trypsin, suggest that limited proteolysis of C5 can produce a chemoattractant molecule without release of free C5a.

Animals↗

PubMed1982