Biomedical subjects

E J Brown

Publications and source records attributed to E J Brown.

At least 19 recordsLinked to original sources

PubMed1992

Expression of the 50-kDa integrin-associated protein on myeloid cells and erythrocytes.

Integrin-associated protein (IAP) is a 50-kDa intrinsic membrane protein that is involved in signal transduction during neutrophil activation by a variety of Arg-Gly-Asp-containing ligands. However, IAP does not itself directly bind these ligands, which are instead recognized by the leukocyte response integrin (LRI). In fact, IAP is more widely expressed than the LRI and is even on erythrocytes, which express no known integrins. This suggests that IAP may have additional functions besides signal transduction in association with the LRI. In this work we have quantitated IAP expression on several myeloid cells and cell lines, as well as erythrocytes, using a mAb which recognizes this protein. These data show that there are about 10,000 IgG-binding sites on each erythrocyte and 20 times that number on the myeloid cell lines U937 and HL60. Normal PMN and monocytes express about 25,000 IgG-binding sites. There are twice as many Fab'-binding sites on each cell, suggesting that each IgG binds via both Ag-combining sites. Binding data using several mAb to IAP suggest that IAP undergoes a temperature-dependent conformational change. Unlike some integrin receptors, IAP is not stored in a regulated secretory compartment in polymorphonuclear leukocytes (PMN). In addition, there is no evidence for internalization or shedding of IAP upon PMN activation. These data show that IAP is expressed at significant levels on myeloid cells and erythrocytes and that its expression is unaffected by the state of PMN activation.

Amino Acid Sequence↗

PubMed1992

Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. The SAVE Investigators.

BACKGROUND: Left ventricular dilatation and dysfunction after myocardial infarction are major predictors of death. In experimental and clinical studies, longterm therapy with the angiotensin-converting--enzyme inhibitor captopril attenuated ventricular dilatation and remodeling. We investigated whether captopril could reduce morbidity and mortality in patients with left ventricular dysfunction after a myocardial infarction. METHODS: Within 3 to 16 days after myocardial infarction, 2231 patients with ejection fractions of 40 percent or less but without overt heart failure or symptoms of myocardial ischemia were randomly assigned to receive doubleblind treatment with either placebo (1116 patients) or captopril (1115 patients) and were followed for an average of 42 months. RESULTS: Mortality from all causes was significantly reduced in the captopril group (228 deaths, or 20 percent) as compared with the placebo group (275 deaths, or 25 percent); the reduction in risk was 19 percent (95 percent confidence interval, 3 to 32 percent; P = 0.019). In addition, the incidence of both fatal and nonfatal major cardiovascular events was consistently reduced in the captopril group. The reduction in risk was 21 percent (95 percent confidence interval, 5 to 35 percent; P = 0.014) for death from cardiovascular causes, 37 percent (95 percent confidence interval, 20 to 50 percent; P less than 0.001) for the development of severe heart failure, 22 percent (95 percent confidence interval, 4 to 37 percent; P = 0.019) for congestive heart failure requiring hospitalization, and 25 percent (95 percent confidence interval, 5 to 40 percent; P = 0.015) for recurrent myocardial infarction. CONCLUSIONS: In patients with asymptomatic left ventricular dysfunction after myocardial infarction, long-term administration of captopril was associated with an improvement in survival and reduced morbidity and mortality due to major cardiovascular events. These benefits were observed in patients who received thrombolytic therapy, aspirin, or beta-blockers, as well as those who did not, suggesting that treatment with captopril leads to additional improvement in outcome among selected survivors of myocardial infarction.

Adult↗

PubMed1992

Phospholipase activation during monocyte adherence and spreading.

Phospholipase activation is an important element in cellular signal transduction. In our study we investigated the role and regulation of phospholipase activation during human monocyte adherence and spreading. In human monocytes, phospholipase inhibition (with bromophenacyl bromide (BPB) or manoalide) impaired cell adherence and spreading. In contrast, neither cyclooxygenase/lipoxygenase inhibition nor platelet activating factor receptor blockade affected these responses. The impaired adherence and spreading induced by phospholipase inhibition with BPB could be partially reversed by the addition of nM levels of arachidonate (20:4(n - 6)). Dihomogammalinolenic acid (20:3(n - 6)) could substitute for arachidonate, but other polyunsaturated fatty acids were ineffective in this regard. The phospholipase inhibitor, BPB was selective in its effects on cellular phospholipase activities. BPB inhibited adherence/spreading-related and PMA-stimulated phospholipase activities, but not Ca2+ ionophore-stimulated phospholipase activity. To further probe for the role of Ca2+ in monocyte adherence and spreading, monocytes were loaded with MAPTAM (bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N', tetraacetic acid tetraacetoxymethyl ester), an EGTA analog. In contrast to phospholipase inhibition, intracellular Ca2+ chelation with MAPTAM did not affect monocyte adherence but did inhibit monocyte spreading. MAPTAM partially inhibited adherence/spreading-stimulated phospholipase activity, but did not inhibit PMA-stimulated phospholipase activity. These data suggest that human monocyte adherence and spreading may sequentially activate Ca(2+)-independent and then Ca(2+)-dependent phospholipases to release arachidonate. The activation of phospholipase and the release of arachidonate appear to be integral parts of the adhesion process.

Acetophenones↗

PubMed1992

Ligand binding specificity of the leukocyte response integrin expressed by human neutrophils.

The ligand binding specificity of the leukocyte response integrin (LRI) expressed by polymorphonuclear leukocyte (PMN) was investigated by examining its interaction with two adhesion motifs within fibrinogen: the alpha chain sequence RGD and the gamma chain sequence KQAGDV. The effect of the hexapeptides KQAGDV, KQRGDV, and KGAGDV on fibrinogen-stimulated phagocytosis, a LRI-dependent function, was examined. Surprisingly, the sequence KGAGDV was most potent for inhibition of fibrinogen-stimulated ingestion; the order of potency of these peptides was KGAGDV greater than KQAGDV greater KQRGDV = GRGDSPA. Latex spheres coated with multivalent KGAGDV bound specifically to PMN and antibodies that recognized either the LRI beta chain (7G2) or an associated protein (IAP)-abrogated bead binding. Various control and anti-beta 1 and anti-beta 2 antibodies did not affect bead binding. Monovalent peptides KGAGDV and KQRGDV were equipotent for inhibition of bead binding to unstimulated PMN (ID50 = 19 microM). In contrast, KGAGDV was more potent than KQRGDV for inhibition of bead binding to N-formylmethionylleucylphenylalanine-stimulated PMN (ID50 = 2.5 microM versus ID50 = 60 microM). A control peptide, KGALEVA, did not inhibit LRI ligand binding or function. These data suggest that the unique amino acid sequence KGAGDV may represent a specific ligand for LRI and that LRI ligand binding specificity may be regulated by the activation state of the cell.

Amino Acid Sequence↗

PubMed1992

Immunoglobulin-mediated phagocytosis by human monocytes requires protein kinase C activation. Evidence for protein kinase C translocation to phagosomes.

This study has investigated the role of protein kinase C (PKC) activation in IgG-mediated phagocytosis by human monocytes. Incubation of monocytes with IgG-opsonized targets increased membrane-associated PKC approximately 2-fold. Kinetic studies showed that the translocation of PKC to membrane occurred before significant ingestion took place. The pharmacologic PKC inhibitor H7 inhibited IgG-dependent ingestion with ID50 of 20 microM, while the structurally related isoquinoline sulfonamide HA1004 had no effect at this concentration. Staurosporine and calphostin C, PKC inhibitors which have different mechanisms of actions than H7, also inhibited ingestion. Depletion of PKC by prolonged incubation with phorbol esters also inhibited phagocytosis, and dose-response curves showed a strong correlation between the extent of PKC depletion and the extent of inhibition of ingestion. Finally, phagosomes were isolated by sucrose density centrifugation of cells disrupted 5 min after the initiation of phagocytosis. Measurement of PKC activity and immunoreactivity in the phagosomes showed that PKC was concentrated in the phagosome membrane approximately 5-fold compared to the uninvolved plasma membrane. Together, these data suggest that PKC activation is an early, essential step in the efficient ingestion of IgG-opsonized targets by monocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Induction and peak gene expression of insulin-like growth factor II follow that of myogenin during differentiation of BC3H-1 muscle cells.

Acting through hormonal and/or autocrine/paracrine mechanisms, the insulin-like growth factors (IGFs) stimulate the differentiation of muscle cells. Previous studies have suggested that one mechanism by which IGFs stimulate muscle cell differentiation is by increasing the expression of myogenin, a DNA binding protein that regulates the expression of muscle-specific genes. While exogenous IGF peptides increase myogenin mRNA, the role of endogenously produced IGF peptides in myogenin expression has not been established. In addition, the potential role of IGFs in regulating the expression of Id, a protein in myoblasts that can inhibit the action of myogenin-like peptides, is also unknown. In the present study, we have examined the kinetics of accumulation of myogenin and IGF-II mRNAs during differentiation of BC3H-1 mouse muscle cells and have explored the potential role of IGFs in regulating Id expression. During differentiation induced by serum withdrawal, induction of myogenin expression preceded that of IGF-II, the principal IGF peptide expressed by these cells. In addition, Id expression decreased within two hours in serum-free medium and was not affected by IGF treatment. Thus, these studies suggest that endogenously-produced IGF-II may stimulate muscle cell differentiation after both the decrease in Id and the induction of myogenin gene expression have occurred.

Animals↗

PubMed1992

Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of intracytoplasmic calcium concentration rise from inositol 1,4,5-trisphosphate.

The signal transduction mechanisms involved in the regulation of phagocytosis are largely unknown. We have recently shown that in neutrophils, when IgG-mediated phagocytosis is stimulated by formyl-methionyl-leucyl-phenyl-alanine (fMLP), the enhanced ingestion is dependent on the increase in [Ca2+]i which results from ligation of Fc receptors by the IgG-coated target (Rosales, C., and Brown, E. (1991) J. Immunol. 146, 3937-3944). Now, we have studied the mechanism by which this rise in [Ca2+]i occurs. Aggregated IgG, the monoclonal antibody 3G8 (which recognizes Fc receptor type III), and insoluble immune complexes caused an increase in [Ca2+]i. The rise in [Ca2+]i induced by Fc receptor ligation was resistant to pertussis toxin. In contrast, fMLP induced a rise in [Ca2+]i which was inhibited by pertussis toxin. fMLP-induced [Ca2+]i was accompanied by an accumulation of inositol 1,4,5-trisphosphate (IP3) which peaked by 15 s, and which was also abolished by pertussis toxin. IP3 accumulation after aggregated IgG, 3G8, or insoluble immune complexes was much less than after fMLP. Unlike [Ca2+]i rise induced by Fc receptor ligation, this small increase in IP3 was inhibited by pertussis toxin. These data demonstrated that the [Ca2+]i increase induced by Fc receptor ligation is not mediated by IP3. Immediate pretreatment of human polymorphonuclear neutrophils with optimal doses of fMLP also reduced subsequent increase in [Ca2+]i rise from thapsigargin, a sesquiterpene lactone tumor promoter that releases intracellular Ca2+ from IP3-sensitive stores without IP3 turnover. Similarly, to its effects on thapsigargin, fMLP inhibited the [Ca2+]i rise upon subsequent immune complex binding. Pretreatment of cells with immune complexes also prevented subsequent [Ca2+]i rise from thapsigargin and fMLP. These data demonstrate that IgG Fc receptor ligation and fMLP activation of human polymorphonuclear neutrophils use distinct signal transduction mechanisms to release Ca2+ from the same thapsigargin-sensitive intracellular pool. In contrast to fMLP, signal transduction for increased [Ca2+]i after Fc receptor stimulation does not involve a pertussis toxin-sensitive G protein, and is independent of IP3.

Adult↗

PubMed1992

Calcium channel blockers nifedipine and diltiazem inhibit Ca2+ release from intracellular stores in neutrophils.

We have undertaken a detailed study of the mechanisms of maintenance of intracellular Ca2+ homeostasis in human polymorphonuclear neutrophils (PMN) and its implications for phagocytosis and IgG Fc receptor (FcR) signaling. When PMN were incubated in Ca(2+)-free medium, cytoplasmic calcium concentration ([Ca2+]i) was markedly depressed and intracellular stores were depleted of calcium. [Ca2+]i in these depleted cells increased within 1 min when PMN were placed in medium containing Ca2+ and then decreased to a level close to the normal basal [Ca2+]i, replenishing the intracellular Ca2+ pools. LaCl3 prevented entry of Ca2+ into Ca(2+)-depleted PMN, but the calcium channel blockers nifedipine, diltiazem, and verapamil did not. Nifedipine and diltiazem but not verapamil inhibited the movement of Ca2+ from cytosol to intracellular stores. Nifedipine and diltiazem inhibited the normal increase in [Ca2+]i from aggregated IgG binding to FcR and also prevented formyl-methionyl-leucyl-phenyl-alanine (fMLP)-induced [Ca2+]i rise. Verapamil had no effect on either an fMLP- or IgG-mediated increase in [Ca2+]i. Consistent with this, nifedipine and diltiazem inhibited fMLP-stimulated phagocytosis (which is dependent on an increase in [Ca2+]i) when PMN had repleted intracellular stores. In contrast, LaCl3 inhibited fMLP-stimulated ingestion only in PMN which had intracellular store depleted. None of these compounds had any effect on phorbol dibutyrate-stimulated ingestion (which is independent of a [Ca2+]i rise). In summary, these data show that Ca2+ is in rapid equilibrium between intracellular and extracellular compartments in PMN. Exchange of cytoplasmic Ca2+ with the extracellular space is inhibited by LaCl3, while exchange of Ca2+ between the cytosol and intracellular stores is inhibited by the dihydropyridine nifedipine and the benzothiazepine diltiazem. These data suggest that these drugs, which are known to regulate some plasma membrane Ca2+ channels in excitable cells, can also regulate Ca2+ release from intracellular stores in PMN and that this regulation may have significant effects on PMN function.

Adult↗

PubMed1992

A monoclonal antibody to the T-cell receptor increases IGF-I receptor content in normal T-lymphocytes: comparison with phytohemagglutinin.

The biological effects of the IGFs are mediated through interaction with specific cell surface receptors. It has been previously reported that mitogenic activation of T-lymphocytes by phytohemagglutinin (PHA) is associated with increased IGF-I receptor content. However, the mechanisms which regulate IGF-I receptor expression during T-lymphocyte activation are unknown. To explore further the regulation of IGF-I receptor expression in T-cells, we investigated IGF-I receptor content and mRNA abundance in T-lymphocytes after stimulation either by PHA or OKT-3, the latter being a monoclonal antibody directed against the CD-3 antigen of the T-cell receptor. IGF-I binding in T-cells demonstrated increased IGF-I receptor content after stimulation by both PHA and OKT-3. Peak binding was induced after 72 h of treatment with PHA and 48 h of treatment with OKT-3. Affinity cross-linking of 125I-IGF-I to T-cell membranes demonstrated a single approximately 130 kDa band which was increased after treatment with PHA or OKT-3. This band was inhibited by the addition of alpha-IR3, a monoclonal antibody to the IGF-I receptor. Both PHA and OKT-3 increased IGF-I receptor mRNA abundance with peak increases at 20 h and 60 h, respectively. Parallel increases in IGF-I receptor and beta-actin mRNA abundance were observed, consistent with previous studies demonstrating increased actin gene expression after T-cell activation. Thus, the increase in IGF-I receptor mRNA abundance markedly preceded the increase in IGF-I receptor content after PHA stimulation, but the increase in IGF-I receptor mRNA abundance followed the increase in IGF-I receptor content after OKT-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1992

Oxygen therapy in sickle cell disease.

The effect of oxygen therapy on the number of irreversibly (ISC) and reversibly (RSC) sickled cells was studied in patients with sickle cell anemia. Inhalation of 50% oxygen in patients who were not in crisis produced a significant fall in RSCs and a lesser fall in ISCs. Twenty-five subjects in sickle cell crisis were chosen at random to receive either oxygen (15 patients) or air (10 patients). Those who received oxygen showed a significant reduction in RSCs, but not in ISCs. No significant change in RSCs or ISCs occurred in the group who received air. Despite the reduction in RSCs in the oxygen-treated group, there was no significant difference between the air and oxygen groups in the duration of severe pain, opioid administration, and hospitalization. It was also observed that crisis was associated with arterial desaturation and a reduction in the number of RSCs. We conclude that, although RSCs may be involved in the pathophysiology of sickle cell crisis, reduction in RSCs by oxygen therapy in these studies did not result in any reduction in the duration of crisis.

Adolescent↗

PubMed1992

Expression and modulation of the vitronectin receptor on human dermal microvascular endothelial cells.

Microvascular endothelial cells express a variety of cell-surface integrins in vivo and in vitro with varying affinities for matrix proteins. The vitronectin receptor (VnR), a complex of the alpha v and beta 3 integrin chains, is capable of binding to a variety of matrix proteins that are deposited in injured tissues, including vitronectin, fibrinogen, and thrombin. Staining of frozen sections of human skin with antibodies recognizing the VnR and examination by immunofluorescence microscopy demonstrates staining in a vascular pattern suggesting in vivo expression of the vitronectin receptor on endothelial cells. Examination of pure cultures of human dermal microvascular endothelial cells (HDMEC) by flow-cytometric analysis and enzyme-linked immunosorbent assay confirmed that HDMEC also express cell surface VnR complex in vitro. Stimulation of human dermal microvascular endothelial cells in vitro with agents that stimulate protein kinase C resulted in dose- and time-dependent increases in expression of alpha v and beta 3 integrin chains. Additionally, stimulation with basic fibroblast growth factor induced similar increases, but stimulation with transforming growth factor-beta or interleukin-1 alpha failed to increase VnR expression. Increases in cell-surface VnR expression also correlated with an increased ability of microvascular endothelial cells to bind to vitronectin, but not fibronectin-coated surfaces. Although increases in cell-surface expression of beta 3 paralleled increases in expression of cell-surface alpha v, regulation of mRNA expression was distinct for each chain. These data suggests that microvascular endothelial cells express the VnR complex in vivo, that the cell-surface expression of this integrin on dermal microvascular endothelial cells can be regulated, and that this regulation may be important in cell adherence, cell migration, and wound healing.

Blotting, Northern↗

PubMed1992

Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin.

Entactin is an integral component of basement membranes that plays a major role in basement membrane assembly through its ability to bind avidly to both laminin and type IV collagen. Because neutrophil (PMN) interactions with entactin have not been examined, we investigated the ability of natural and recombinant entactin to mediate PMN adhesion and chemotaxis. With both forms of entactin, we observed that entactin-coated surfaces promoted PMN adhesion and that entactin stimulated PMN chemotaxis. The increase in adhesion to entactin over control was two to threefold whereas the chemotactic response to 15 ng/ml (1 x 10(-10) M) entactin was equivalent to the chemotactic response elicited with 1 x 10(-8) M formyl-methionyl-leucyl-phenylalanine (fMLP). HL-60 cells, after differentiation with dimethylsulfoxide, also demonstrated adhesion and chemotaxis to entactin. A synthetic peptide of the Arg-Gly-Asp (RGD) domain in entactin, SIGFRGDGQTC (S-RGD), mediated PMN adhesion and chemotaxis, and preexposure of PMN to S-RGD blocked PMN adhesion and chemotaxis induced by entactin without diminishing the adhesive and chemotactic activities of fMLP. In contrast, preexposure to peptides SIGFRGEGQTCA or SIGFKGDGQTCA had no effect. The findings with synthetic peptides were confirmed with a recombinant entactin mutant in which aspartic acid at residue 674 was replaced with glutamic acid, thus converting the RGD sequence of entactin to RGE. RGE-entactin was neither adhesive nor chemotactic for neutrophils. Monoclonal antibodies to the leukocyte response integrin (LRI) and the integrin-associated protein blocked entactin-mediated adhesion and chemotaxis whereas monoclonal antibodies to beta 1 and beta 2 integrins had no effect and PMN from an individual with leukocyte-adhesion deficiency adhered normally to entactin-coated surfaces. These data demonstrate that entactin mediates biologically and pathologically important functions of PMN through its RGD domain and that LRI, which has been shown previously to mediate RGD-stimulated phagocytosis, is also capable of mediating RGD-stimulated PMN adhesion and chemotaxis.

Amino Acid Sequence↗

PubMed1992

Complement receptors, adhesion, and phagocytosis.

Phagocytosis is an essential component of host defense against invading pathogens. Although many cell types can ingest particulate material, "professional phagocytes" perform this function much more efficiently than other cells because they express unique membrane proteins and signal transduction mechanisms. This review addresses the current state of knowledge about the process of phagocytosis and its regulation.

Animals↗

PubMed1991

Arachidonic acid is essential for IgG Fc receptor-mediated phagocytosis by human monocytes.

Phagocytosis is a specialized function of neutrophils and macrophages that requires coordination of multiple biochemical and biophysical events. Considerable progress has been made in identifying the membrane receptors involved in phagocytosis, but the intracellular signaling pathways that are necessary for particle ingestion are poorly understood. In an effort to address this complex question, we investigated the role of arachidonic acid (AA) in the uptake of yeast and IgG-coated E (EIgG) or C-coated E. Human monocytes, labeled with 3H AA, released this label during phagocytosis of yeast and EIgG, but not in response to EC3b. The PL inhibitors bromophenacyl bromide and manoalide abolished the release of 3H and inhibited phagocytosis of EIgG in parallel. Both drugs caused a similar inhibition of yeast-mediated 3H release but had little effect on yeast ingestion. Similar results were obtained with the inhibitor quinacrine (mepacrine). Exogenously added AA and dihomo-gamma-linolenic acid restored bromophenacyl bromide-inhibited EIgG ingestion; arachidonate analogs eicosatrienoic acid and eicosapentanoic acid did not. Inhibition of the cyclooxygenase and lipoxygenase pathways for AA metabolism by indomethacin or BW755C did not affect EIgG phagocytosis, demonstrating that these major AA metabolic pathways are not involved in phagocytic signaling. These experiments suggest that release of AA is essential for EIgG ingestion and that phagocytosis in monocytes proceeds by at least two mechanisms, one dependent on AA (EIgG) and one independent of it (yeast).

Acetophenones↗

PubMed1991

Modulation of fibronectin-mediated Bacillus Calmette-Guérin attachment to murine bladder mucosa by drugs influencing the coagulation pathways.

Adjuvant intravesical Bacillus Calmette-Guérin (BCG) has proved to be an effective treatment for superficial bladder cancer. Intraluminal attachment of BCG organisms via binding to the extracellular matrix protein, fibronectin (FN), appears to be required for expression of the antitumor efficacy of BCG against a murine bladder tumor. Initial studies demonstrated that radiolabeled FN localized to the acutely injured urothelium but not to intact urothelium. These studies also demonstrated that exogenous administration of FN enhanced BCG attachment to the injured but not to the intact urothelium. Because FN has been shown to be an integral part of clot formation at sites of urothelial injury, drugs known to affect fibrin clot formation were tested for their effects on BCG attachment and antitumor efficacy in a murine bladder tumor model. A stabilizer of fibrin clot formation was shown to enhance both BCG attachment and antitumor efficacy in the same model. An increased number of BCG organisms were also retained in the lymph nodes and spleens of mice receiving fibrin clot stabilizers, suggesting indirectly that immunological mechanisms are involved in the antitumor efficacy of BCG. The data presented herein provide further support for the hypothesis that BCG attachment to the injured bladder is mediated by FN. Furthermore, modulation of BCG-FN attachment is demonstrated to be possible with drugs influencing the coagulation pathway. This attachment is shown to be required for the antitumor efficacy in a murine bladder tumor model, and thus modulation of BCG-FN attachment appears to have significant influence on the antitumor efficacy of BCG in the murine bladder tumor model.

Aminocaproic Acid↗

PubMed1991

Two mechanisms for IgG Fc-receptor-mediated phagocytosis by human neutrophils.

Neutrophils (PMN) stimulated with the chemo-attractant FMLP, with platelet activating factor (PAF), and with phorbol dibutyrate exhibited a three- to fivefold increase in phagocytosis of IgG-opsonized sheep E (ElgG). Enhancement of phagocytosis occurred even when stimulants were washed away before the ElgG were added, showing that they could prime PMN for enhanced phagocytosis. When PMN were loaded with MAPTAM, a cell permeant analog of EGTA that prevented any rise in [Ca2+]i, they showed no FMLP-stimulated phagocytosis but had a normal phagocytic response to PAF and phorbol dibutyrate. Addition of MAPTAM after FMLP priming abolished enhanced ingestion, even in the presence of optimal extracellular Ca2+. Thus, the [Ca2+]i rise that occurred on ligation of PMN IgG FcR by ElgG was required for FMLP-stimulated phagocytosis. We determined which FcR were involved in this [Ca2+]i rise and in FMLP-stimulated phagocytosis. Aggregated (agg-IgG) IgG and the anti FcRIII mAb 3G8 both caused increases in [Ca2+]i, removal of FcRIII with phosphatidylinositol-specific phospholipase C (PIPLC) abolished the 3G8-dependent rise in [Ca2+]i without affecting the agg-IgG-dependent rise. Moreover, IV.3 (anti FcRII mAb) completely inhibited the agg-IgG-induced increase in [Ca2+]i in PIPLC-treated PMN, showing that ligation of FcRII is sufficient for a normal IgG-induced [Ca2+]i rise. FMLP-stimulated phagocytosis also was unaffected by PIPLC, suggesting that the rise in [Ca2+]i required for FMLP-stimulated phagocytosis could come from FcRII ligation. From these studies we conclude that there are two molecular mechanisms for IgG-mediated phagocytosis in activated PMN. One, stimulated by FMLP, is dependent on an increase of intracellular Ca2+ during the ingestion process; the other, activated by phorbol esters and PAF, is capable of effecting high levels of ingestion at very low concentrations of [Ca2+]i. These data are consistent with the hypothesis that ligation of FcRII by IgG opsonized targets is sufficient for this Ca(2+)-dependent mechanism of stimulated phagocytosis.

Calcium↗

PubMed1991

Intracellular signaling events associated with the induction of proliferation of normal human B lymphocytes by two different antigenically related human B cell growth factors (high molecular weight B cell growth factor (HMW-BCGF) and the complement factor Bb).

High molecular weight B cell growth factor (HMW-BCGF) and the complement component, Factor B, are antigenically related. HMW-BCGF and the physiologic Factor B activation fragment Bb, are both mitogenic for B lymphocytes and compete for binding to the B cell plasma membrane (Peters, M., Ambrus, J. L., Jr., Fauci, A., and Brown, E. (1988) J. Exp. Med. 168, 1225-1235). To understand which second messengers that occur after ligand-receptor interaction are associated with mitogenesis, we have examined the early signaling events after stimulation of activated B cells with these related growth factors. HMW-BCGF but not Bb increased [cAMP]i with a maximum between 45 and 60 min after stimulation. The increase in [cAMP]i was inhibited by indomethacin, suggesting that prostaglandin synthesis is involved in this response. Increase in [cAMP]i induced by HMW-BCGF, cholera toxin, or dibutyryl cAMP was associated with increased expression of the HMW-BCGF receptor, but there was no increase in proliferation of activated B cells when they were stimulated with cAMP agonists other than HMW-BCGF. These data suggest that cAMP is associated with regulation of receptor expression but is neither necessary nor sufficient for induction of proliferation. Both HMW-BCGF and Bb increased cellular levels of diacylglycerol and a water-soluble molecule which could be labeled with both [3H]myoinositol and [14C] glucosamine. However, only HMW-BCGF induced increases in intracellular calcium. Thus, two antigenically related B cell growth factors, HMW-BCGF and Bb, produce overlapping but distinct sets of second messengers after incubation with Sac-activated B cells. Since both induced increases in diacylglycerol and water-soluble inositol, one or both of these molecules may be involved in the proliferative signal generated by the related growth factors. In contrast, the increase in [cAMP]i caused by HMW-BCGF but not Bb is involved in the signal to increase HMW-BCGF receptor expression, but is unrelated to proliferation.

B-Lymphocytes↗

PubMed1991

Cytokine regulation of complement receptor-mediated ingestion by mouse peritoneal macrophages. M-CSF and IL-4 activate phagocytosis by a common mechanism requiring autostimulation by IFN-beta.

Macrophage CSF (M-CSF, CSF-1) and IL-4 are two cytokines known to have effects on mature monocytic phagocytes in vitro. In this report we show that M-CSF and IL-4 activate resident mouse peritoneal macrophages to ingest particles via their C3b and C3bi receptors, which are not capable of mediating ingestion in resting cells. IgG-mediated ingestion was also increased by IL-4 and M-CSF. IL-1, IL-2, TNF-alpha, and IFN-gamma were not able to stimulate C receptor-mediated ingestion. Stimulation by IL-4 and M-CSF is dependent upon high cell density and greater than 24-h exposure to the cytokine. Interestingly, antibody to IFN-alpha/beta and mAb to IFN-beta inhibited the enhanced ingestion caused by both M-CSF and IL-4. However, neither IFN-alpha nor IFN-beta alone stimulated C receptor-mediated ingestion. M-CSF did not affect the ligand-independent distribution of CR3 on the macrophage surface. We conclude that two apparently unrelated cytokines, M-CSF and IL-4, both enhance macrophage phagocytosis of C and IgG-coated targets via a common pathway in which autocrine stimulation with IFN-alpha/beta is necessary but not sufficient.

Animals↗