Depletion of guanine nucleotides with mycophenolic acid suppresses IgE receptor-mediated degranulation in rat basophilic leukemia cells

In RBL-2H3 rat basophilic leukemia cells, Ag that crosslink IgE-receptor complexes stimulate the turnover of inositol phospholipids, the mobilization of Ca2+ from intra- and extracellular sources, the release of serotonin and other substances from granules and the transformation of the cell surface from a microvillous to a lamellar architecture. This study explores the role of GTP-binding proteins (G proteins) in the control of these biochemical and functional responses. We report that incubating RBL-2H3 cells for 4 h with 10 microM mycophenolic acid (MPA), an inhibitor of de novo GTP synthesis, reduces GTP levels by over 60% and causes an average reduction of 50% in Ag-stimulated serotonin release. This inhibition of secretion is associated with a 50% decrease in the rate of 45Ca2+ influx in MPA-treated cells. In contrast, Ag-stimulated inositol trisphosphate production is only slightly reduced, indicating that the phosphatidylinositol-specific phospholipase C can be activated by Ag in GTP-depleted cells. The membrane responses to IgE receptor cross-linking are unaffected by incubating cells with MPA. Exogenous guanine or guanosine protects the GTP pools in MPA-treated cells and permits normal ion transport and secretory responses to Ag; adenine does not. These results implicate a guanine nucleotide-binding protein in the control of IgE receptor-dependent signal transduction in RBL-2H3 cells. This protein may particularly control the Ca2+ influx pathway that is essential for secretion.     

Characterization of the target cell receptor for IgE. II. Polyacrylamide gel analysis of the surface IgE receptor from normal rat mast cells and from rat basophilic leukemia cells.

Purified rat peritoneal mast cells (RMC) and cultured rat basophilic leukemia (RBL) cells were surface labeled with 125I by using lactoperoxidase, incubated with unlabeled rat monoclonal IgE and subjected to solubilization by treatment with Nonidet P-40 (NP-40). With both cell types significant amounts of radioiodinated material could be specifically precipitated by a "sandwich" system consisting of rabbit anti-rat epsilon-chain and goat anti-rabbit Ig. The precipitates were dissociated with sodium dodecyl sulfate (SDS) and urea and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. With RMC three radioactive bands were seen. One corresponded to IgE present on the RMC at the time of isolation. A small band migrating in the region of light chain was seen with both sepcific (anti-IgE) and control precipitates. It showed no demonstrable relationship to IgE. The major radioactive band corresponded to a m.w. of 62,000. This band was dependent upon the presence of IgE and was not found when non-IgE binding control cells were used. With RBL cells, only the IgE-dependent 62,000 dalton peak was present. Saturation of the IgE receptor sites of the RMC or RBL cells before lactoperoxidase labeling almost totally eliminated this radioactive band, indicating that cell-bound IgE rendered this membrane component inaccessible to the radiolabel. These results strongly suggest that this cellular component is identical, at least in part, with the target cell surface receptor for reaginic antibody. The data also further support the hypothesis that the neoplastic RBL cells have a normal surface receptor for IgE.     

Characterization of an antiserum specific for cell surface antigens of rat mast cells.

Rabbits were immunized with rat peritoneal mast cells (RMC) in complete Freund's adjuvant. The antisera (anti-RMC) were checked for their reactivity with RMC by intradermal skin tests in rats. The best serum was selected and absorbed with rat liver cells and rat immunoglobulins, including IgE. The absorbed serum (anti-RMCabs), as well as the anti-RMC serum, were then tested for their reactivity with RMC. Both sera were cytotoxic to RMC but only anti-RMC was cytotoxic for rat lymph node cells. Both sera gave positive reactions in rat skin, as seen by the permeability to Evan's blue dye. The binding of rat IgE to RMC was also inhibited by both sera. A control rabbit anti-rat sarcoma serum absorbed with liver cells did not show any interaction with RMC. When 125I-labeled RMC surface antigens were precipitated with anti-RMCabs and analyzed by SDS polyacrylamide gel electrophoresis, several components were observed. Among these was one with a mobility identical to that of a mast cell surface component that had previously been identified as the receptor for IgE or at least a component thereof.     

Involvement of hrs binding protein in IgE receptor-triggered exocytosis in RBL-2H3 mast cells

Hrs binding protein (Hbp) tightly associated with Hrs is thought to play a regulatory role in vesicular trafficking during endocytosis and exocytosis. In this study, we have expressed dominant-negative mutants of Hbp to evaluate their effects on the degranulation of secretory granules in RBL-2H3 mast cells. The dominant-negative mutants of Hbp significantly inhibited IgE receptor (FcepsilonRI)-triggered secretory response as tested by beta-hexosaminidase release. These results suggest that Hbp functions as a regulator in the FcepsilonRI-triggered degranulation of secretory granules in mast cells.     

Kinetics of fusion of the cytoplasmic granules with phagocytic vacuoles in human polymorphonuclear leukocytes. Biochemical and morphological studies

This study on human neutrophils was conducted to measure the kinetics of degranulation of the different cytoplasmic granules into phagocytic vacuoles, and to relate the timing of these events to the burst of respiration that accompanies phagocytosis by these cells. Purified neutrophils were incubated with latex particles opsonized with human immunoglobulin (Ig)G, and phagocytosis was stopped at timed intervals. The cells were examined by electron microscopy to document the sequence of degranulation of the cytoplasmic granules. The azurophil granules and lyosomes were identified by histochemical staining for peroxidase and acid phosphatase, respectively. Phagocytic vacuoles were separated from cell homogenates by floatation on sucrose gradients and assayed for contained lactoferrin, myeloperoxidase, and acid hydrolases. The conclusions drawn from the biochemical and morphological studies were in agreement and indicated: particle uptake and vacuole closure can be completed within 20 s; both the specific and azurophil granules fuse with the phagocytic vacuole much earlier than is generally appreciated, with half-saturation times of 39 s (99% confidence limits, 15-72); oxygen consumption has kinetics similar to those of the fusion of these granules with the phagosome; degranulation of the acid hydrolases beta- glucuronidase, N-acetyl-beta-glucosaminidase (biochemical assays), and acid phosphatase (biochemical assay and electron microscopic cytochemistry) have kinetics of degranulation that are similar to each other but totally different from and much slower than that of myeloperoxidase with half-saturation times of between 354 and 682 s (99% confidence limits, 246-883). This suggests that the acid hydrolases are not co-located with myeloperoxidase in the azurophil granule but are contained in distinct lysosomes, or "tertiary granules".     

Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay.

BACKGROUND: Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-alpha, and leukotrienes. METHODS: Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme beta-hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay. RESULTS: Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and beta-hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein. CONCLUSIONS: The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis.     

Differential regulation of neutrophil phospholipase d activity and degranulation

One of the proposed functions of phosphatidic acid (PA) formation from phospholipase D (PLD) activation in neutrophils is to promote degranulation induced by receptor agonists. The present study shows that the time course and dose response of PA formation and degranulation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) differed. PLD activation and degranulation also exhibited different dose response to genistein and epigallocatechin gallate (EGCG), inhibitors of protein tyrosine kinases. Genistein inhibited PLD activity with an IC(50) value of 12.2 microM in fMLP- and 107 microM in phorbol myristate acetate (PMA)-stimulated cells. It required higher concentrations of genistein to inhibit degranulation than to inhibit PLD activity induced by fMLP. EGCG in the range of 40-400 microM had no effect on PLD activity but it inhibited the release of beta-glucuronidase and elastase by fMLP-stimulated cells. These results demonstrate differential regulation of PLD activity and degranulation of primary granules by genistein and EGCG in fMLP-stimulated neutrophils.     

Antibody and inhibitor of chymase inhibit histamine release in immunoglobulin E-activated mast cells

The low-molecular-weight inhibitor of chymase, chymostatin, and F(ab')2 fragments of anti-chymase markedly inhibited histamine release induced by anti-rat immunoglobulin E (IgE) but not that induced by compound 48/80. Inhibitors with molecular weights of more than 6,000, such as alpha 1-antichymotrypsin and aprotinin, and non-immunized F(ab')2 had no effect on histamine release. These results suggest that chymase in mast cell granules plays an essential role in the process of IgE-mediated degranulation. After degranulation, released chymase was associated with the cell surface while released tryptase was present in the extracellular milieu as a complex with a protein associated with tryptase (trypstatin).     

Membrane-bound IgE receptor complexes fused with rat basophilic leukemia cells mediate degranulation

The high affinity receptor for IgE on rat basophilic leukemia (RBL) cells mediates antigen-triggered cellular degranulation. Polyethylene glycol-induced membrane fusion methods were used to introduce exogenous IgE receptors into living RBL cells, and these were tested for normal activities. In cell-cell fusion experiments, RBL cells with fluorescein-labeled rat IgE bound to receptors and containing [5-1,2-3H(N)]hydroxytryptamine binoxalate ([3H]5HT) in their secretory granules were fused to cells with receptors occupied by rhodamine-labeled anti-dinitrophenyl mouse IgE. The fused cells showed a uniform surface distribution of both types of IgE, which could be patched independently by anti-IgE or dinitrophenylated bovine gamma globulin (DNP16BGG). [3H]5HT release could be triggered specifically by DNP16BGG. In vesicle-cell fusion experiments, plasma membrane vesicles, with receptors occupied by fluorescein- and 125I-labeled anti-DNP mouse IgE, were fused to RBL cells containing [3H]5HT. The cells showed substantial associated fluorescein fluorescence and 125I counts, and [3H]5HT release could be triggered specifically by DNP16BGG. These experiments indicate that IgE receptors can be dissociated from their natural cellular interactions and retain the ability to reassociate with another cell's components to deliver the transmembrane signal for degranulation.     

Characterization of the stimulatory effect of medroxyprogesterone acetate and chlormadinone acetate on growth factor treated normal human breast epithelial cells

OBJECTIVE: Evidence is increasing that adding progestogens to hormone replacement therapy may be more harmful than beneficial, however it is debatable whether all progestogens act equally on breast cells. Mitogenic growth factors from stromal breast tissue are important in growth-regulation of breast cells, and may modify responses to progestogens. We investigated the effect of two C-21 derivatives, medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) on growth-factor treated normal breast epithelial cells and tried to explore the underlying mechanisms of proliferation. METHOD: MCF10A (human epithelial, estrogen- and progesterone-receptor negative normal breast cells) were incubated with MPA or CMA at 0.1 and 1 microM for 7 days with the growth factors (GFs) EGF, bFGF and IGF-I at 1pM. The same combinations, as well as growth factors alone, were also incubated with the proliferation inhibitors PD98059 and LY294002 at 1 microM for 4 days. Cell proliferation rate was measured by the ATP-assay. RESULTS: MPA 0.1 and 1 microM, and CMA 1 microM in combination with GFs both significantly increased cell proliferation rate, with MPA having the greatest effect. MPA- and CMA-induced proliferation of GF stimulated cells was blocked by both PD98059 (selective inhibitor of MAP kinases) and LY294002 (phosphatidylinositol 3-kinase inhibitor); GF stimulated cells could not be significantly reduced by any of the inhibitors used. CONCLUSION: MPA and CMA have a stimulatory effect on benign growth factor stimulated MCF10A cells, possibly via activation of MAP kinase and subsequent substrates and activation of PI3-kinase. GF induced proliferation appear to be mediated by pathways other than those investigated here. Growth factors and progestogens therefore have an additive, synergistic effect on cell proliferation, eliciting their effects via different pathways.     

Cell cycle specific induction of HL-60 cell differentiation and apoptosis by mycophenolic acid

HL-60 cells undergo terminal differentiation and apoptosis in response to different types of sub-toxic and toxic perturbations respectively. The mechanism by which cells sense different amounts of perturbation to activate pathways that lead to the engagement of a relevant biological response is not known. The response of HL-60 cells to treatment with the immunosuppressant mycophenolic acid (MPA), a specific inhibitor of dGTP/GTP-synthesis, allowed quantitation of a metabolic perturbation which triggered a cellular response. 1.5 microM MPA induced 38% terminal differentiation to CD14 positive, early monocyte-like cells and 22% cell death by apoptosis, whereas 3 microM MPA induced 70% apoptosis but no differentiation. Despite the difference in biological outcomes, 72 h exposure to both 1.5 microM and 3 microM MPA caused a similar ( approximately 75%) depletion of total GTP levels. Cells synchronized by centrifugal elutriation were treated with MPA. Elutriated cells were overall less sensitive to the effects of MPA but 3 microM MPA induced significantly less apoptosis and more differentiation in an elutriation-enriched G1-population than in a population normally distributed in the cell cycle, suggesting that the effects of MPA in S-phase may subsequently lead to cell death. However, analysis of apoptosis by using a terminal deoxynucleotidyltransferase assay and measurement of bromodeoxyuridine incorporation showed that apoptosis was engaged in G1. Analysis of the phosphorylation status of the retinoblastoma protein demonstrated that Rb was hypophosphorylated prior to apoptosis and that in apoptotic cells, separated by flow cytometry, Rb protein was absent, presumably due to proteolysis. The loss of Rb protein did not appear to permit transit to S-phase, and was not accompanied by an expression of c-Myc. Surprisingly, therefore, an antimetabolite inducing a loss of GTP brought about cell death by apoptosis in the G1 phase of the cell cycle.     

Novel apoptosis assay to analyze immunosuppression.

BACKGROUND: We developed a novel whole blood assay to assess the effects of immunosuppressants on lymphocyte apoptosis. METHODS: Peripheral blood for six experiments for each drug was treated either with cyclosporin A (CsA), mycophenolate acid (MPA), tacrolimus (TRL) or rapamycin (RAPA). Whole blood was stimulated with different concentrations of staurosporine (0-5 microM) for 24 h. Using FACS, apoptosis were measured by Annexin V expression (%cells +/- SEM). RESULTS: MPA, RAPA, TRL, but not CsA, increased (P < 0.05) apoptotic cells (MPA:20.9 +/- 3.7; RAPA:17.5 +/- 3.3; TRL:16.4 +/- 2.9 vs. control:15.2 +/- 2.8). CONCLUSION: This new whole blood assay exhibited that MPA has a stronger effect on apoptosis than RAPA and TRL, whereas CsA had no effect.     

Stimulation of degranulation from human eosinophils by platelet-activating factor

Platelet-activating factor (PAF) is a highly active mediator which has been implicated in allergic inflammation and bronchial asthma, possibly by interacting with eosinophils. We have examined the effect of PAF on activation of purified human eosinophils as measured by degranulation (eosinophil peroxidase, eosinophil cationic protein, arylsulfatase B, beta-glucuronidase, and alkaline phosphatase) and oxidative metabolism (superoxide anion production). PAF induced enzyme release at concentrations ranging from 1 pM to 10 microM in a rapid (t1/2 5 to 8 min), Ca2+-dependent and noncytotoxic manner from both the specific and small granules, whereas its biologic precursor and metabolite, lyso-PAF, had no effect. For all enzymes, maximal enzyme release occurred at 100 nM PAF with a mean ED50 value of 1.47 +/- 0.4 nM. At this concentration the mean percentage of total enzyme release by PAF from specific granules was 20.3 +/- 1.6% (17.9% for eosinophil peroxidase, 20.6% for beta-glucuronidase, 22.4% for alkaline phosphatase) and 28.8 +/- 2.2% from small granules (arylsulfatase B). Calcium ionophore A23187, PMA, and opsonized zymosan also induced eosinophil degranulation but their peak effect after 10-min incubation with maximal release 14.7%, 12.9%, or 14.1%, respectively, was lower when compared with PAF. Incubation of eosinophils with the PAF-antagonist WEB 2086 led to a parallel shift of the dose-response curve to the right, indicating a competitive antagonism. PAF also caused generation of superoxide anions by human eosinophils but this occurred at higher concentrations of PAF (1 microM to 30 microM) with an ED50 of 8.4 +/- 0.9 microM. Again, this effect was competitively inhibited by WEB 2086. These studies demonstrate that PAF activates human eosinophils to release granule constituents and generate superoxide anions. Since both PAF and eosinophil products are associated with pathogenesis of bronchial asthma our findings may be of particular pathophysiologic relevance.     

Potential role of mycophenolate mofetil in the management of neuroblastoma patients

In human neuroblastoma cell lines (LAN5, SHEP and IMR32), mycophenolic acid (MPA) at concentrations (10(-7)-10(-6) M) readily attainable during immunosuppressive therapy with mycophenolate mofetil (Cellcept), induces guanine nucleotide depletion leading to cell cycle arrest and apoptosis through a p53 mediated pathway (up-regulation of p53, p21 and bax and down-regulation of bcl-2 and survivin). MPA-induced apoptosis is also associated to a marked decrease of p27 protein. In the same cell lines MPA, at lower concentrations (50 nM), corresponding to the plasma levels of the active free drug during Cellcept therapy, induces differentiation toward the neuronal phenotype by causing a partial chronic guanine nucleotide depletion. MPA-induced differentiation is not associated to p27 accumulation as occurs using retinoic acid. At a fixed concentration of MPA a higher percentage of apoptotic or differentiated cells is obtained when non dialysed serum substitutes for the dialysed one, due to the higher hypoxanthine concentration in the former (about 10 microM) leading to competition on HPRT-mediated salvage of guanine. At hypoxanthine or oxypurinol concentrations higher than 1 microM (up to 100 microM) no further enhancement of MPA effects was obtained, in agreement with the recently described safety of the allopurinol-mycophenolate mofetil combination in the treatment of hyperuricemia of kidney transplant recipients. The apoptotic effects of MPA do not appear to be significantly increased by the UDP-glucuronosyltransferase inhibitor niflumic acid.     

Mast cell membrane antigens and Fc receptors in anaphylaxis: II. Functionally distinct receptors for IgG and for IgE on mouse mast cells

The present work was aimed at analyzing the functional relationships between mouse mast cell receptors for IgG and IgE antibodies. It was based on a study of the inhibition of IgG1-and IgE-induced passive mast cell degranulation produced by various immunoglobulin preparations capable of interfering with Fc receptors. Rat myeloma IgE, a high-affinity ligand for IgE receptors, was used to search for a possible participation of IgE receptors in IgG1-dependent degranulation. Mouse myeloma IgG, which inhibited only weakly IgG1-mediated reactions, had no chance to compete successfully with high-affinity IgE antibodies, but aggregated HGG was found to behave as a high-affinity ligand for IgG receptors. This enabled us to search for a possible participation of IgG receptors in IgE-dependent degranulation. The results show that rat myeloma IgE and aggregated HGG specifically inhibited IgE-induced and IgG1-induced reactions, respectively, but failed to inhibit reactions not requiring free Fc receptors. The conclusion was that receptors for IgG and for IgE are functionally independent on mouse mast cells, and are both expressed on the same cells.     

Differential up-regulation of specific and azurophilic granule membrane markers in electropermeabilized neutrophils.

We have developed an alternative method to study the degranulation in electropermeabilized human neutrophils by measuring the up-regulation of the specific membrane markers CD63 (residing in the azurophilic granules of resting neutrophils) and CD67 (present in specific granules). The expression of these marker proteins was measured by the binding of specific antibodies to paraformaldehyde-fixed cells and subsequent flow cytometry. We first investigated whether the changes in CD63 and CD67 expression after stimulation of intact cells were comparable with earlier measurements of neutrophil degranulation, in which the release of soluble marker proteins was measured. These experiments indicated that this new method compares favourably with earlier studies, both with respect to kinetics and stimulus dependency. Subsequently, we applied this method (which does not include centrifugation of the cells) to study degranulation in electropermeabilized neutrophils. In permeabilized neutrophils, a clear up-regulation of the specific granule marker CD67 was observed upon incubation with a free Ca2+ concentration of 1 microM, a value of the cytosolic free Ca2+ concentration occurring in formylmethionyl-leucyl-phenylalanine (FMLP)-activated neutrophils. The azurophilic granule marker CD63 required GTP-gamma-S besides 1 microM Ca2+ for a significant up-regulation. Hence, our study indicates a different requirement for intracellular signals of the two main types of granules in human neutrophils.     

ELECTRON MICROSCOPE OBSERVATIONS ON COMPOUND 48/80-INDUCED DEGRANULATION IN RAT MAST CELLS : Evidence for Sequential Exocytosis of Storage Granules

In vitro degranulation of rat mast cells was studied at different intervals ranging from 10 to 60 sec after adding the histamine liberator, compound 48/80 (0.4 µg/ml, 17°C). The ultrastructural changes were followed by electron microscopy, and parallel assays were made to determine the histamine released. In addition, the extracellular tracers lanthanum and hemoglobin (demonstrated by its peroxidative activity) were applied to mast cells to follow communication of the extracellular space with the cavities formed during degranulation. After a lag period of 10 sec, degranulation started in the most peripherally located granules. The perigranular membrane fused with the plasma membrane, resulting in a pore bridged by a thin diaphragm. This was followed by rupture of the diaphragm and extrusion of the granule matrix (exocytosis). The process advanced towards the cell interior by fusion and opening of the deeper situated granules to the formerly opened granule cavities. At the end of the process, the cell was filled by a system of complicated cavities containing a number of altered granules. Extracellular tracers have shown that these intracellular cavities were in unbroken communication with the extracellular space from the very beginning of their formation. Both lanthanum and hemoglobin were found to be adsorbed to the limiting membrane of the cavities and bound to altered mast cell granules. In contrast, no tracer substance was present in nondegranulating mast cells. Degranulation of mast cells by compound 48/80 is regarded as a sequential exocytosis, a process similar to that described for some exocrine gland cells. All the "intracellular" cavities, formed by degranulation, were shown to communicate with the extracellular space; consequently, granules lying in these cavities must be considered as biologically extracellular. The present findings support the view that histamine is released from the granule matrix by the extracellular ionic milieu.     

Degradation of human anaphylatoxin C3a by rat peritoneal mast cells: a role for the secretory granule enzyme chymase and heparin proteoglycan

Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0 degrees C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared with 37 degrees C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with bis (sulfosuccinimidyl) suberate (BS3) covalently cross-linked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC by using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan.     

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.