Localized mast cell degranulation induced by concanavalin A-sepharose beads. Implications for the Ca2+ hypothesis of stimulus-secretion coupling

Concanavalin A (Con A) covalently linked to Sepharose 4B beads induced localized degranulation of sensitized rat peritoneal mast cells in regions of contact between beads and cells. This degranulation was Ca2+ dependent and was not seen when sensitized mast cells bound to beads conjugated with a nonstimulating lectin, wheat germ agglutinin, or when unsensitized mast cells bound to Con A-Sepharose. The finding that sensitized mast cells which had adhered to Con A-Sepharose beads degranulated in regions of the cell away from the area of bead contact if exposed to soluble Con A excluded the possibility that the localized release was due to a redistribution of the IgE receptors or putative Ca2+ channels to the region of bead contact. The results suggest that, if an influx of Ca2+ is the mechanism for initiating mast cell degranulation, then the opening of Ca2+ channels in the plasma membrane of activated mast cells is a localized event and that Ca2+ acts locally within the cell to initiate exocytosis.     

A new immunochemical method for the quantitative measurement of specific gene products in man-rodent somatic cell hybrids

Summary An immunochemical method has been developed for the quantitative determination of species-specific gene products, for instance a-galactosidase and N-acetyl-a-galactosaminidase, in man-rodent hybrid cells and in the parental cell lines. Antisera raised against the purified enzymes are covalently coupled to Sepharose 4B. The gene products are specifically removed from a cell lysate by incubating with the appropriate Sepharose-coupled antiserum. After centrifugation followed by washing of the precipitated Sepharose, the enzymic activity can be quantitatively measured on the Sepharose beads. With this technique it has been demonstrated that the ability of human N-acetyl-a-galactosaminidase (also known as a-galactosidase B) to hydrolyze a-galactosidic linkages is lost when the enzyme is expressed in man-Chinese hamster hybrid cells.     

Functional binding analysis of human fibrinogen as an iron- and heme-binding protein

Human fibrinogen is a metal ion-binding protein, but its mechanism of binding with iron and heme has not been elucidated in detail. In this study, human fibrinogen was immobilized on CNBr-activated Sepharose 4B beads. The fibrinogen beads bound hemin (iron–protoporphyrin IX: PPIX) as well as iron ion released from ferrous ammonium sulfate (FAS) more efficiently than Sepharose 4B beads alone. Hemin bound to fibrinogen still exhibited pseudo-peroxidase activity. The affinity of fibrinogen binding to hemin, Sn–PPIX, Zn–PPIX and metal-free PPIX followed the order Sn–PPIX < metal-free PPIX < hemin < Zn–PPIX; PPIX bound more non-specifically to control beads. FAS significantly enhanced the binding of hemin to fibrinogen beads. These results suggest that human fibrinogen directly recognizes iron ion, the PPIX ring and metal ions complexed with the PPIX ring, and that the binding of hemin is augmented by iron ions.     

The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides

Phosphorothioate oligodeoxynucleotides (sODN) containing the CpG motif or TCG repeats induce T cell-independent polyclonal activation of human B cells. To elucidate the mechanism of this response, the role of cell surface receptors was investigated. Sepharose beads coated with stimulatory but not nonstimulatory sODNs induced B cell proliferation comparably with soluble sODNs. The B cell stimulatory activity of Sepharose-bound sODN did not result from free sODN released from the beads since media incubated with coated beads were inactive. Using FITC-labeled sODNs as probes, binding to human B cells could be detected by flow cytometry. Binding was rapid, saturable, initially temperature independent, but with a rapid off-rate. Competition studies indicated that both stimulatory sODNs and minimally stimulatory sODNs bound to the same receptor. By contrast, phosphodiester oligonucleotides with the same nucleotide sequence as sODNs and bacterial DNA inhibited the binding of sODNs to B cells minimally. Charge appeared to contribute to the binding of sODNs to B cells since binding of sODNs was competitively inhibited by negatively charged molecules, including fucoidan, poly I, and polyvinyl sulfate. These data indicate that human B cells bind sODNs by a receptor-mediated mechanism that is necessary but not sufficient for polyclonal activation.     

Antigen-induced proliferation and immunoglobulin A secretion by a human-human hybridoma

The capacity of membrane immunoglobulin A (IgA)-bearing B cells to respond to specific antigen in the absence of T cell influences has not been defined. A human-human hybridoma, constructed from an Epstein-Barr virus transformed tonsil B cell that secreted IgA anti-phosphorycholine (PC) and a human plasmacytoma cell, was utilized to examine this issue. The cloned hybridoma expressed membrane IgA and secreted IgA specific for PC. Stimulation of the hybridoma cells with PC conjugated to Sepharose beads (PC-Sepharose) but not glycine-conjugated Sepharose resulted in an increase in DNA synthesis. Affinity purified goat anti-human IgA bound to Sepharose also augmented DNA synthesis. Soluble PC did not increase DNA synthesis and inhibited the increase in DNA synthesis resulting from PC-Sepharose. IgA secretion was augmented in response to PC-Sepharose, as demonstrated by an increase in the number of Ig-secreting cells detected by a reverse hemolytic plaque assay and by quantitation of the IgA secreted per cell by enzyme-linked immunosorbent assay. Mitogen-stimulated T cell supernatants increased IgA secretion of the hybridoma cells but did not cause synergistic stimulation of the cells in the presence of PC-Sepharose. These data indicate that Sepharose-bound antigen was sufficient to induce proliferation and augment IgA secretion by this membrane IgA anti-PC-bearing hybridoma. The results suggest that cross-linking of membrane IgA by specific antigen may be a sufficient stimulus for proliferation and differentiation of B cells at this stage of maturation.     

Iron-dependent binding of bovine milk α-casein with holo-lactoferrin, but not holo-transferrin

Bovine milk α-casein has been identified as an iron- and heme-binding protein. However, the physiological role of its iron-binding remains to be elucidated in more detail. α-Casein was immobilized on CNBr-activated Sepharose 4B beads, and the α-casein agarose beads efficiently bound hemin as well as ferrous ammonium sulfate (Fe(2+)) as compared with control beads. Additionally, α-casein-beads bound bovine holo-lactoferrin (Lf), but not holo-transferrin. Lf caused the release of Fe(2+) which had bound to the α-casein-agarose beads beforehand. These results suggest that bovine α-casein iron-dependently binds holo-bovine Lf more strongly than Fe(2+), and that strong binding between them may play a physiological role in regulating iron homeostasis in the bovine mammary gland.     

Characterization of a concanavalin A supernatant-derived idiotype-specific T helper cell factor

We have previously demonstrated the requirement of two T helper (Th) populations for the expression of plaque-forming cells (PFC) that bear the dominant cross-reactive idiotype (CRI) associated with the phenyltrimethylammonium (TMA) response (1). In addition to the classic major histocompatibility complex-restricted Th cell, the response was also dependent upon the so-called second order Th2 population, which binds to idiotypic determinants, is carrier specific, but does not require hapten linked to carrier for function. This cell type can be replaced by supernatant (Sn) media from concanavalin A (Con A)-stimulated naive spleen cells. This report involves the study of the Con A Sn derived factor(s) responsible for the expression of CRI bearing PFC populations. When the Brucella abortus (BA)-trinitrophenol (TNP) conjugated antigen is added to TNP-ovalbumin-primed A/J-derived spleen cells in culture, anti-TNP PFC are generated of which only less than or equal to 5% bear the CRI normally associated with anti-TMA antibodies. Upon addition of Con A Sn, the total number of generated anti-TNP PFC doubles, whereas the percentage and number of CRI+ PFC increases approximately eightfold to 10-fold. The factor(s) responsible for this activity are T cell derived, bear Jk serologic determinants, and can be detected in the Sn as early as 4 hr after Con A stimulation. The material appears to be late acting, because it can augment the CRI+ anti-TNP response when added as late as 24 hr before termination of the cultures. In addition, the factor(s) can be bound to and eluted from CRI+ anti-TMA and anti-TNP monoclonal antibodies coupled to Sepharose 4B beads, but not from their CRI- counterparts (i.e., CRI- anti-TMA and anti-TNP antibodies), nor from A/J normal mouse immunoglobulin-coupled beads. Most interestingly, the factor(s) also bind to and can be eluted from the TMA ligand coupled to Sepharose 4B, but not from TNP-Sepharose conjugates. All of these results are consistent with the support the contention that the factor(s) is derived from a Th2-like subpopulation. As assayed by standard protocols, the isolated material contains no T cell replacing factor, interleukin 2, or B cell growth factor activity.     

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.     

Independent effects of IgG and complement upon human polymorphonuclear leukocyte function.

Particle ingestion by polymorphonuclear leukocytes (PMN) is promoted by cell surface recognition and binding of fragments of the third component of complement (C3) and Fc regions of certain immunoglobulin (IgG) molecules. In order to determine the influence of these specific ligandsurface membrane interactions upon other PMN functions, we have employed nonphagocytosable particles (serum-treated Sepharose beads) coated with fragments of C3 and/or IgG, and have investigated whether these provide a sufficient stimulus for the metabolic changes and degranulation that ordinarly accompany phagocytosis by PMN. Sepharose 4B activates complement in fresh normal serum and consequently is coated with fragments of C3 (confirmed by immunoelectrophoretic evidence of factor B and C3 conversion and by immunofluorescence). Adsorbed IgG could be removed from serum-treated Sepharose by boiling in 2 M NaCl without significantly influencing bound complement. We have found that normal human PMN recognize and adhere to Sepharose beads coated with fragments of C3 and consequently are stimulated to increase their oxidative metabolism (measured as superoxide anion generation). This PMN response occurred in the absence of IgG but could be amplified if this immunoglobulin was also present on the bead surfaces.Both adherence and metabolic stimulation could be blocked by treatment of the beads with F(ab)2 anti-C3. In contrast to metabolic stimulation, degranulation (selective extracellular release of lysosomal constituents) was observed only when PMN encountered both C3 fragments and IgG on the beads. This response could be blocked by treating beads with either F(ab)2 anti-C3 or F(ab)2 anti-IgG. These results indicate that cell surface stimulation of PMN is not an "all or none" phenomenon and that certain vital functions of these cells may be mediated or modulated independently by immunoglobulins and complement.     

The role of Ras guanine nucleotide releasing protein 4 in Fc epsilonRI-mediated signaling, mast cell function, and T cell development

The RasGRP (Ras guanine nucleotide-releasing protein) family proteins are guanine nucleotide exchange factors that activate Ras GTPases, ultimately leading to MAPK activation and many cellular processes. The RasGRP family has four members. Published studies demonstrate that RasGRP1, RasGRP2, and RasGRP3 play critical roles in T cells, platelets, and B cells, respectively. RasGRP4 is highly expressed in mast cells. Although previous data suggest that it is important in mast cell development and function, the role of RasGRP4 in mast cells and allergic responses has not been clearly demonstrated. In this study, we generated RasGRP4(-/-) mice to examine the function of RasGRP4. Analyses of these mice showed that mast cells were able to develop normally in vivo and in vitro. Despite high levels of RasGRP4 expression in mast cells, RasGRP4 deficiency led to only a modest reduction in FcεRI-mediated degranulation and cytokine production. Interestingly, mast cells deficient in both RasGRP1 and RasGRP4 had a much more severe block in FcεRI-mediated signaling and mast cell function. We also made the unexpected finding that RasGRP4 functions during thymocyte development. Our data suggest that after the engagement of immunoreceptors, immune cells likely employ multiple members of the RasGRP family to transduce critical signals.     

Plucking of lectin receptors from erythrocytes: Isolation of cell surface components without the use of detergents

A new approach is described for the isolation of lectin receptors without the use of detergents, by plucking them from the cell surface. Cells bound to lectin-coated Sepharose beads are sheared off the beads by mechanical disruption, whereupon the receptors remain attached to the beads and are released specifically by inhibitory sugars. Material plucked from neuraminidase-treated human erythrocytes by beads coated with peanut agglutinin and released by D-galactose was identified as asialoglycophorin. The same membrane glycoprotein was plucked from neuraminidase-treated erythrocytes by beads coated with soybean agglutinin, but at considerably lower yield.     

Binding of stimulated human peripheral blood lymphocytes to Sepharose-concanavalin A is not reversed by methyl-α-mannoside

Concanavalin A (con A) bound to Sepharose beads stimulates human peripheral blood lymphocytes and as with soluble con A, DNA synthesis can be prevented by the addition of methyl-α-mannoside (MAM) 1 hr after exposure to mitogen. In contrast to the response of cells stimulated with soluble con A, addition of MAM 24 hr after the start of incubation with Sepharose bound mitogen does not prevent a second round of DNA replication as determined in bromodeoxyuridine density transfer experiments indicating that MAM does not dissociate the Sepharose-con A responding cell complex. We infer that within 24 hr, lymphocytes develop associations with con A-Sepharose beads at non MAM dissociable sites.     

Purification and analysis of the core protein of the protease-resistant intracellular chondroitin sulfate E proteoglycan from the interleukin 3-dependent mouse mast cell

Chondroitin sulfate E proteoglycan was extracted in the presence of protease inhibitors from 6 X 10(9) mouse bone marrow-derived, interleukin 3-dependent mast cells, of which 3 X 10(7) had been biosynthetically labeled with [35S]sulfate or [3H]glycine. Chondroitin sulfate E proteoglycan was purified to apparent homogeneity by density-gradient centrifugation, differential molecular weight dialysis, DEAE-52 ion exchange chromatography, and Sepharose CL-4B gel filtration chromatography. Chondroitin sulfate E proteoglycan, radiolabeled with [3H]glycine or [35S]sulfate, filtered as a single peak of radioactivity on Sepharose CL-4B with a Kav of 0.41. When purified [3H]glycine-labeled proteoglycan was digested with chondroitinase ABC and subjected to gel filtration, all of the radioactivity was shifted to a lower molecular weight. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr of the peptide core obtained by chondroitinase ABC treatment was approximately 10,000. The purified proteoglycan was resistant to degradation by collagenase, clostripain, trypsin, chymotrypsin, elastase, chymopapain, V8 protease, proteinase K, and Pronase, as assessed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the core peptide of the intact proteoglycan revealed that glycine, serine, and glutamic acid/glutamine accounted for 70% of the total amino acids and were present in a molar ratio of 4.3/1.6/1.0. When analyzed for neutral hexose content by gas-liquid chromatography, the proteoglycan contained approximately 2% of its weight as mannose, fucose, galactose, and other sugars, indicating that oligosaccharides were linked to the peptide core. The mouse bone marrow-derived mast cell chondroitin sulfate E proteoglycan, like the rat serosal mast cell heparin proteoglycan, is markedly protease resistant, has highly sulfated glycosaminoglycans, and contains a peptide core that is rich in serine and glycine. These characteristics of the mast cell class of intracellular proteoglycans may contribute to their function in stimulus-induced granule secretion as well as in mediator storage, including retention of cationic neutral proteases.     

Biochemical characterization of a mast cell plasma membrane antigen shared by mouse serosal, culture-derived, and virally transformed mast cells

The expression of the antigenic determinant identified by the B54.2 rat monoclonal antibody on four populations of mouse mast cells has been quantified, and the epitope-bearing surface antigen and its biosynthesis have been characterized. As assessed by indirect immunofluorescence staining and flow cytometric analysis, B54.2 antibody bound to serosal mast cells (S-MC), bone marrow culture-derived mast cells (BM-MC), fetal liver culture-derived mast cells (FTL-MC), and Abelson murine leukemia virus-transformed FTL-MC (ABFTL-MC). However, the intensity of cell surface fluorescence exhibited by ABFTL-MC was approximately eightfold less per cell compared with nontransformed, culture-derived mast cells. Immunoprecipitation of B54.2 antibody-binding molecules from each population of mast cells labeled intrinsically with [35S]methionine and analysis by SDS-PAGE demonstrated that the B54.2 epitope was expressed in each case on two noncovalently associated proteins of 110,000 Mr and approximately 130,000 Mr, but that the percentage of radiolabel in the latter species was approximately threefold less in ABFTL-MC than in BM-MC. As assessed by pulse-chase analysis with [35S]methionine, the 110,000 Mr protein was a precursor of the 130,000 Mr molecule ("B54.2 antigen") synthesized by BM-MC. Labeling of BM-MC with [35S]methionine in the presence of tunicamycin followed by immunoprecipitation and SDS-PAGE of B54.2 antibody-binding material revealed a single species of 93,000 Mr, indicating that the native molecules contained N-linked carbohydrate. Endoglycosidase H treatment of the glycoproteins precipitated by B54.2 antibody from BM-MC reduced the Mr of the 110,000-Mr molecule to 93,000 Mr without an appreciable change in the 130,000-Mr species. These data indicate that the 110,000-Mr precursor form is a "high mannose" type glycoprotein and the 130,000-Mr membrane surface B54.2 antigen is a "complex" type glycoprotein, and that the epitope recognized by the B54.2 antibody on the surface of the mouse mast cell populations is located on the 93,000-Mr peptide core.     

De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line

It is an accepted hypothesis that the nerve growth factor protein (NGF) plays an important role in the development of vertebrate sympathetic and sensory ganglia and has effects on some central neurons. The best known NGF species is that isolated from the mouse submaxillary gland, MSG-NGF. MSG-NGF can be isolated as a subunit containing protein, 7S-NGF, made up of three dissimilar subunits called alpha-, beta-, and gamma-NGF. Beta-NGF is the biologically active subunit and its synthesis in vivo and in vitro has been demonstrated. Less is known about the synthesis of the alpha- and gamma-NGF or the assembly of the subunits into the 7S complex. In order to develop a clonal model system for the study of NGF synthesis, processing and secretion, affinity chromatography techniques were applied to cell extracts of S180 mouse sarcoma, a cell line known to synthesize NGF. After incubating S180 cells in³⁵S-Methionine, cell extracts were exposed to antibody directed against alpha-NGF, gamma-NGF or beta-NGF covalently bound to Sepharose beads in order to elute and characterize the desired NGF subunits. Parallel experiments using immunoabsorbed [³⁵S]Methionine-beta-NGF were carried out in the presence or absence of excess NGF, in order to demonstrate the specificity of this procedure. Affinity chromatography with a substrate analogue to arginine ester bound to Sepharose beads was also used to isolate de novo synthesized gamma-NGF. We were able to show that the S180 line synthesized alpha-, beta-, and gamma-NGF indistiguishable from alpha-, beta-, and gamma-NGF isolated from mouse submaxillary gland in terms of antigenic and physicochemical properties, and biological and enzymatic activities. These results are consistent with the hypothesis that NGF is synthesized, assembled and secreted by a single cell type.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Glycosaminoglycan profiles in cloned granulated lymphocytes with natural killer function and in cultured mast cells: their potential use as biochemical markers

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.     

Regulation of the inflammatory response in asthma by mast cell products

In airways, mast cells lie adjacent to nerves, blood vessels and lymphatics, which highlights their pivotal importance in regulating allergic inflammatory processes. In asthma, mast cells are predominantly activated by IgE receptor cross linking. In response to activation, preformed mediators that are stored bound to proteoglycans, for example, TNF-alpha, IL-4, IL-13, histamine, tryptase and chymase, are released. New synthesis of arachidonic acid metabolites (leukotriene C4 (LTC4), leukotriene B4 (LTB4) and prostaglandin D2 (PGD2)) and further cytokines is stimulated. Mediators from degranulating mast cells are critical to the pathology of the asthmatic lung. Mast cell proteases stimulate tissue remodelling, neuropeptide inactivation and enhanced mucus secretion. Histamine stimulates smooth muscle cell contraction, vasodilatation and increased venular permeability and further mucus secretion. Histamine induces IL-16 production by CD8+ cells and airway epithelial cells; IL-16 is an important early chemotactic factor for CD4+ lymphocytes. LTC4, LTB4 and PGD2 affect venular permeability and can regulate the activation of immune cells. The best characterized mast cell cytokine in asthmatic inflammation is TNF-alpha, which induces adhesion molecules on endothelial cells and subsequent transmigration of inflammatory leucocytes. IL-13 is critical to development of allergic asthma, although its mode of action is less clear.     

Fc epsilon RI-mediated association of 6-micron beads with RBL-2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling

Cells of the mucosal mast cell line, RBL-2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (Fc epsilon RI) by soluble cross-linking ligands. This cellular degranulation process requires sustained elevation of cytoplasmic Ca2+. In this study, we investigated the response of RBL-2H3 cells to 6- micron beads coated with IgE-specific ligands. These ligand-coated beads cause only small, transient Ca2+ responses, even though the same ligands added in soluble form cause larger, more sustained Ca2+ responses. The ligand-coated 6-micron beads also fail to stimulate significant degranulation of RBL-2H3 cells, whereas much larger ligand- coated Sepharose beads stimulate ample degranulation. Confocal fluorescence microscopy shows that the 6-micron beads (but not the Sepharose beads) are phagocytosed by RBL-2H3 cells and that, beginning with the initial stages of bead engulfment, there is exclusion of many plasma membrane components from the 6-micron bead/cell interface, including p53/56lyn and several other markers for detergent-resistant membrane domains, as well as an integrin and unliganded IgE-Fc epsilon RI. The fluorescent lipid probe DiIC16 is a marker for the membrane domains that is excluded from the cell/bead interface, whereas a structural analogue, fast DiI, which differs from DiIC16 by the presence of unsaturated acyl chains, is not substantially excluded from the interface. None of these components are excluded from the interface of RBL-2H3 cells and the large Sepharose beads. Additional confocal microscopy analysis indicates that microfilaments are involved in the exclusion of plasma membrane components from the cell/bead interface. These results suggest that initiation of phagocytosis diverts normal signaling pathways in a cytoskeleton-driven membrane clearance process that alters the physiological response of the cells.     

Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation

We used a morphometric and autoradiographic approach to analyze changes in specific cytoplasmic granules and cytoplasmic lipid bodies associated with human lung mast cell degranulation. Mast cells were dissociated from lung tissue by enzymatic digestion and were then enriched to purities of up to 99% by countercurrent centrifugation elutriation and recovery from columns containing specific antigen bound to Sepharose 6 MB. Degranulation was induced by goat anti-IgE. At various intervals after stimulation, parallel aliquots of cells were recovered for determination of histamine release or were fixed for transmission electron microscopy. We found that lipid bodies, electron- dense structures that lack unit membranes, were present in both control and stimulated mast cells. Autoradiographic analysis showed that lipid bodies represented the major repository of 3H-label derived from [3H]arachidonic acid taken up from the external milieu. By contrast, the specific cytoplasmic granules contained no detectable 3H-label. In addition, lipid bodies occurred in intimate association with degranulation channels during mast cell activation, but the total volume of lipid bodies did not change during the 20 min after stimulation with anti-IgE. This result stands in striking contrast to the behavior of specific cytoplasmic granules, the great majority of which (77% according to aggregate volume) exhibited ultrastructural alterations during the first 20 min of mast cell activation. These observations establish that mast cell cytoplasmic granules and cytoplasmic lipid bodies are distinct organelles that differ in ultrastructure, biochemistry, and behavior during mast cell activation.     

Mast cells: an unexpected finding in the modulation of cutaneous wound repair by charged beads

Increased numbers of mast cells are affiliated with a broad spectrum of pathologic skin conditions, including ulcers, atopic dermatitis, neurofibromatosis, hemangiomas, keloids, and hypertrophic scars. It has been proposed that mast cells play a primary pathophysiologic role in these disorders and that their presence represents not merely a secondary event. While investigating their recent hypothesis that positively charged cross-linked diethylaminoethyl dextran (CLDD) beads potentiate cutaneous wound healing, the authors serendipitously observed increased numbers of mast cells in the deep dermis of wounds treated with CLDD beads. The authors propose that mast cells may play an important role in the modulation of healing seen with CLDD beads. Incisional wounds were studied in 30 Sprague-Dawley rats partitioned into two groups that were killed 7 or 14 days after wounding. The wounds were treated with positively, negatively, or neutrally charged CLDD beads. Physiologic saline served as a control. At the designated times after incisional wounding, biopsy specimens were tested for wound breaking strength or processed for histologic testing, fixed in 4% paraformaldehyde, and stained with Giemsa and Goldner-Masson trichrome. Mast cells were counted under light microscopy in a blinded fashion and were expressed as the number of cells per millimeter squared. Significant increases in the number of mast cells were observed in the deep dermis of incisional wounds after implantation with positively or negatively charged CLDD beads. In contrast, neutrally charged beads had no effect on mast cell numbers. At 7 days, the incisions treated with positively charged beads averaged 2.1 times more mast cells compared with those treated with physiologic saline or neutrally charged beads, whereas the incisions treated with negatively charged beads displayed 3.2 times more mast cells. By day 14, the incisions treated with positively charged beads averaged 2.5 times more mast cells than those wounds treated with saline or neutrally charged beads; the incisions treated with negatively charged CLDD beads had 3.4 times more mast cells. The 7-day tensiometric data indicated that wounds treated with negatively charged CLDD beads had increased breaking strength compared with wounds treated with neutrally charged beads or saline (1.8 and 1.7 times, respectively; p = 0.01 and p = 0.02). Wounds treated with positively charged beads also showed increased breaking strength compared with wounds treated with neutrally charged beads or saline (1.5 and 1.4 times greater); however, this did not reach statistical significance. There was no apparent difference in breaking strength when neutrally charged beads were compared with those treated with saline. At 14 days, there was no statistically significant difference in wound breaking strength between different treatments. These findings are clinically germane to the assessment of proposed therapeutic applications of CLDD beads for a variety of impaired wound-healing states. Furthermore, if increased mast cell populations are intimately linked to hypertrophic scar and keloid formation, the results of the authors' study suggest that CLDD bead therapy of cutaneous wounds may lead to pathologic wound healing in humans.