Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes

Phorbol myristate acetate (PMA) exerts a biphasic effect on receptors for C3b and C3bi of human polymorphonuclear leukocytes (PMN). The addition of PMA for 10 min enhances the capacity of these receptors to promote binding and phagocytosis of C3b- and C3bi-coated erythrocytes. Upon additional incubation for 60 min, the capacity of these receptors to bind and ingest ligand-coated erythrocytes decreases to levels below those of resting cells. Although PMA does cause increased expression of cell surface C3b and C3bi receptors, the sequential rise and fall of receptor activity cannot be accounted for by alterations in the number of surface receptors. It appears, rather, that PMA causes qualitative changes in these receptors, first an increase in receptor activity (activation) and then a decrease in receptor activity (deactivation). In contrast with its effects on C3 receptors, PMA causes only a reduction in the capacity of Fc receptors to bind and ingest IgG-coated erythrocytes. Deactivation of Fc receptors also appears to involve a qualitative alteration in receptors, because the binding affinity of soluble immune complexes is sharply reduced upon stimulation of PMN with PMA. To explore the role of phosphorylation in the sequential activation and deactivation of phagocytosis-promoting receptors, we loaded PMN with thiophosphate (thioP). This compound is incorporated into cellular nucleotides and proteins, and the resultant (thio)phosphorylated proteins are resistant to phosphatases. thioP-loaded cells show enhanced receptor activity, suggesting that activation of receptors is mediated by a phosphorylation event. Cells loaded with thioP and treated with PMA for 70 min do not deactivate C3 or Fc receptors, suggesting that the deactivation is the result of a dephosphorylation event.     

Clustering of ligand on the surface of a particle enhances adhesion to receptor-bearing cells

Human leukocytes express a receptor that mediates the binding of cells and particles coated with C3bi, a fragment of the third component of complement. Previous data indicate that the capacity of this receptor to mediate binding is regulated by changes in its aggregation state. Randomly distributed receptors bind ligand very inefficiently, but stimulation of polymorphonuclear leukocytes with phorbol esters causes a ligand-independent clustering of the receptors in the membrane, and the clustered receptors avidly bind C3bi-coated cells (1). We examined whether the aggregation state of surface-bound ligands also affects the efficiency of binding between receptors and ligands. We found that erythrocytes bearing C3bi in clusters were bound by both macrophages and polymorphonuclear leukocytes far more avidly than erythrocytes bearing the same number of ligands in random array. We made similar observations with erythrocytes coated with C3b, a ligand that is recognized by a separate receptor. Our observations show that the ability of a receptor-bearing cell to bind particles coated with the corresponding ligands is dramatically affected by the distribution of ligand on the surface of the particle. Cell-cell interactions may thus be regulated by alterations in the two-dimensional distribution of receptors and ligands on opposing cell surfaces.     

The interaction of small oligomers of complement 3B (C3B) with phagocytes. High affinity binding and phorbol ester-induced internalization by polymorphonuclear leukocytes

The binding of soluble, multimeric ligands of the major cleavage fragment of complement component 3 (C3b) to polymorphonuclear leukocytes (PMN) has been examined. The oligomers bound entirely via complement C3 receptor type 1 (CR1). There was a single affinity of binding (0.65 nM) at 37 degrees C, while this high affinity binding accounted for only a minority of ligand bound at 0 degree C, with the rest showing a 50-100-fold lower affinity. Azide, fluoride, cytochalasin B, and colchicine had no effect on oligomer binding to PMN. Binding of oligomers had no effect on CR1 expression by PMN. C3b oligomers were not spontaneously internalized by PMN, but were internalized in response to phorbol dibutyrate (PDBu). Both CR1 initially present on the PMN plasma membrane and CR1 initially present in the internal pool of receptors were able to participate in PDBu-induced ligand internalization. C3b oligomers attached to the detergent-insoluble cell cytoskeleton during incubation at 37 degrees C, but cytochalasin B did not inhibit PDBu-induced ligand internalization. Internalized ligand was no longer associated with the detergent-insoluble cytoskeleton. These data demonstrate that 1) some CR1 diffusion is required for optimal oligomer binding; 2) high affinity ligand is not a signal for plasma membrane expression of the internal pool of CR1; 3) CR1 cross-linking is not a sufficient signal for endocytosis; and 4) functional CR1 association with the cytoskeleton which occurs at the plasma membrane is not required for ligand internalization.     

PMA induces the ligand-independent internalization of CR1 on human neutrophils

Phorbol myristate acetate (PMA) has been reported to confer on the C3b receptor (CR1) of neutrophils a capacity for phagocytosis of particles bearing C3b without the involvement of other membrane receptors. In the present study, we employed a monoclonal antibody, YZ-1, that is specific for CR1 to assess the effect of PMA on plasma membrane expression of CR1, total cellular CR1, and internalization of CR1 by neutrophils. PMA had a biphasic effect on the membrane expression of CR1 by purified neutrophils, with 4 ng/ml inducing a 60% increment in receptor expression, and higher concentrations causing up to a 70% decrement. PMA-dependent increases in CR1 expression were not accompanied by corresponding changes in total cellular CR1 and were preempted by treatment of cells with formyl-methionyl-leucyl-phenylalanine (FMLP). PMA-induced decreases in CR1 expression by neutrophils, as measured by binding of indirectly fluoresceinated or radiolabeled YZ-1, or of 125I-labeled dimeric C3b, were maximal with 20 to 30 ng/ml PMA, and occurred within 30 min of incubation at 37 degrees C. The PMA-dependent down-regulation of CR1 by neutrophils was not associated with a comparable decrease in total cellular CR1, and this response was observed to occur also with monocytes but not with peripheral blood lymphocytes. By tagging neutrophil CR1 with 125I-YZ-1 Fab and monitoring accessibility to Protease, intracellular CR1 (inaccessible) was discriminated from receptor on plasma membrane (accessible). Internalization of CR1 occurred within 5 min after addition of PMA to neutrophils, was dose dependent, and involved up to two-thirds of the tagged receptors. Therefore, PMA caused internalization of CR1 by neutrophils in the absence of ligand, indicating that this response was independent of a transmembrane signal generated by a C3b-CR1 interaction.     

Independent regulation of human neutrophil chemotactic receptors after activation

The fluoresceinated chemotactic factors, C5a, formyl-methionyl-leucyl-phenylalanyl-lysine (FMLPL), and casein were used in conjunction with flow cytometry to examine chemotactic factor receptor expression on polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate (PMA), C5a, or formyl-methionyl-leucyl-phenylalanine. Activation with PMA resulted in a dose-dependent increase in binding of fluorescein-labeled (FL)-casein and (FL-FMLPL) over the range of PMA concentrations from 0.5 to 50 ng/ml. In contrast, activation of PMN with PMA resulted in a dose-dependent decrease in FL-C5a binding, and activation with concentrations above 5 ng/ml resulted in a complete loss of binding. This loss of binding was not caused by inactivation of the ligand or prevented by the addition of superoxide dismutase and catalase or protease inhibitors. Furthermore, incubation of PMN with supernatants from PMN stimulated to degranulate did not reduce the availability of C5a receptors. This pattern of increased FMLPL and casein binding with decreased C5a binding was also observed with cytochalasin B-pretreated PMN that were stimulated with chemotactic factors. Parallel studies of superoxide anion generation demonstrated that PMA-treated PMN were still responsive to formyl-methionyl-leucyl-phenylalanine, but not to C5a. These data demonstrate that the activation of PMN up-regulates formyl peptide and casein receptors whereas C5a receptors are down-regulated under similar conditions.     

Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis.

Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN.     

Staurosporine inhibits neutrophil phagocytosis but not iC3b binding mediated by CR3 (CD11b/CD18).

C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data.     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Abstract Porphyromonas gingivalis culture supernate was found to induce hemotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of aglutination induced by the secretagogues PMA and FMLP. Lipopolysaccaharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors of PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity fot P. gingivalis cells but when PMN were treated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutinatin or enhanced adherence could also lead to decreased phagocytic capacity of the adherant or agglutinated cells.     

Dissociation between aggregation and superoxide production in human granulocytes

Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Porphyromonas gingivalis culture supernate was found to induce homotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of agglutination induced by the secretagogues PMA and FMLP. Lipopolysaccharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors on PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity for P. gingivalis cells but when PMN were pretreated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutination or enhanced adherence could also lead to decreased phagocytic capacity of the adherent or agglutinated cells.     

Differential augmentation by recombinant human tumor necrosis factor-alpha of neutrophil responses to particulate zymosan and glucan

Zymosan (Z) and its major insoluble carbohydrate component beta-linked glucan activate human neutrophils (PMN) through a trypsin-sensitive recognition mechanism. This mechanism is believed to involve the PMN CR3R. Both Z and glucan generated dose and time-dependent release of the secondary lysosomal granule marker vitamin B12 binding protein, leukotriene B4 (LTB4) and superoxide from PMN and were phagocytosed with similar dose-dependent kinetics. The PMN superoxide and LTB4 responses to glucan; however, were consistently greater than those to the same doses of Z. The phagocytosis of both particles was significantly reduced after partial digestion with beta-laminarinase but not beta-glucosidase or alpha-mannosidase suggesting a recognition mechanism dependent on intact beta-1,3-glucosidic bonds in both particles. TNF-alpha (rhTNF-alpha) promoted a time- and dose-dependent increase in the expression of PMN CR3 up to 60 min. The increased expression of CR3 was paralleled by the release of the secondary lysosomal granule marker vitamin B12-binding protein. This granule contains a population of CR3R in its boundary membrane and it is the fusion of this membrane with the plasma membrane that may represent the mechanism by which CR3 expression is increased. Preincubation of PMN with 10(-9)M rhTNF-alpha augmented phagocytosis, LTB4, and superoxide generation by PMN in response to activation by Z. In contrast, none of the responses to glucan was significantly increased after incubation with rhTNF-alpha. These differences suggest a lack of absolute homology between the recognition mechanisms for zymosan and glucan and that there is a component of the recognition mechanism for zymosan that is independent of that for glucan and is up-regulated after rhTNF-alpha pretreatment.     

Sodium fluoride reveals multiple pathways for regulation of surface expression of the C3b/C4b receptor (CR1) on human polymorphonuclear leukocytes

We have examined the effects of NaF on C3b receptor (CR1) expression and function in human polymorphonuclear leukocytes (PMN). Plasma membrane expression of CR1 was determined with a monoclonal antibody (3D9); CR1 function was assessed with erythrocytes bearing C3b (EC3b) or C3b oligomers prepared with avidin and biotin. NaF inhibited in a dose-dependent manner CR1-mediated phagocytosis and NaF inhibited f-met-leu-phe or phorbol dibutyrate-induced increases in CR1 expression, with 50% inhibition at 5 mM NaF. Increased plasma membrane expression of CR3 induced by f-met-leu-phe also was inhibited by NaF. However, increased CR1 and CR3 expression due to incubation at 37 degrees C were unaffected by 10 mM NaF. Incubation of PMN with 10 mM NaF depleted 80% of intracellular adenosine triphosphate (ATP) after 10 min. However, inhibition of CR1 function was unrelated to ATP level, inasmuch as normal increases in CR1 expression and in phagocytosis occurred 20 min after removal of NaF, whereas ATP levels remained below 25% of normal. Strikingly, internalization of soluble oligomeric C3b ligands was unaffected by 10 mM NaF, which completely inhibited phorbol dibutyrate-induced CR1 internalization and EC3b phagocytosis. We conclude that there are two different mechanisms for increasing plasma membrane expression of CR1, one of which is inhibitable by NaF. Moreover, there are two distinct pathways of CR1 internalization which can also be distinguished by their sensitivity to NaF.     

Phorbol myristate acetate receptors in human polymorphonuclear neutrophils

Resting or phorbol myristate acetate (PMA)-pretreated neutrophils were disrupted by nitrogen cavitation and were fractionated on Percoll density gradients to identify the subcellular location of PMA receptors. Receptors were found in the cytoplasm of resting cells; neither primary nor secondary granules bound [3H]PMA, and the few binding sites located in non-granule membrane fractions appeared to reflect cytosolic contamination. Contrastingly, PMA-pretreated cells lost cytosolic receptors; greater than 80% of PMA-binding sites were associated with non-granule membranes. Protein kinase C activity similarly shifted from cytosol to membranes after PMA treatment. Indeed, protein kinase C and PMA receptors co-sedimented on Percoll gradients, co-eluted from Ultragel AcA 44 columns loaded with neutrophil cytoplasm, and were identically influenced by various phospholipids. Finally, PMA, mezerein, diacylglycerol, and dialkylglycerol activated protein kinase C with potencies that paralleled their respective abilities to stimulate neutrophil aggregation responses and inhibit [3H]PMA binding to whole cells or cytosol. These results fit a model of stimulus-response coupling wherein exogenous PMA or endogenous diacylglycerol solvate in cellular membranes. Cytosolic protein kinase C binds to the intramembranous ligand, forming an active, membrane-associated complex that phosphorylates nearby elements involved in triggering aggregation and other responses.     

Protein kinase C and phorbol ester receptor expression related to growth and differentiation of nullipotent and pluripotent embryonal carcinoma cells

We have characterized effects of phorbol, 12-myristate 13 acetate (PMA) on growth and differentiation in a nullipotent embryonal carcinoma (EC) cell line, F9, in a pluripotent EC line, P19, and in the differentiated derivatives of these cells, In P19EC and F9EC PMA addition resulted in inhibition of growth, while in the differentiated derivates PMA was mitogenic. PMA did not induce differentiation in EC cells but potentiated the retinoic acid (RA) induced differentiation in P19EC, although, not in F9EC. Rapid morphological changes by PMA were seen in P19EC and two differentiated derivatives which represent different stages of differentiation. In F9 no rapid morphological changes were induced by PMA. Using [3H]phorbol dibutyrate as a ligand we showed that during differentiation into endoderm-like cells the number of phorbol ester receptors increases, while in epithelial-like derivatives no increase is found. In differentiated cells with an increased number of phorbol ester receptors, the cytoplasmic Ca2+- and phospholipid-dependent protein kinase (the putative receptor for phorbol esters) activity was also increased. Only in those derivatives where the number of phorbol ester receptors is increased, is the binding of epidermal growth factor (EGF) inhibited by PMA. These results suggest a relationship between levels of expression of phorbol ester receptors, cytoplasmic protein kinase C and biological effects, namely rapid morphological changes, altered growth, potentiation of RA induced differentiation, and inhibition of EGF binding.     

Translocation of protein kinase C in rat basophilic leukemic cells induced by phorbol ester or by aggregation of IgE receptors

Rat basophilic leukemic cells contain protein kinase C (PKC), 96 +/- 1% of which is located in the cytosol in the resting state. Phorbol ester (PMA), synergistically with calcium ionophore (A23187), caused 55% of the total PKC activity to associate rapidly with membranes where it remained for at least 20 min. When IgE-loaded cells were activated by Ag, maximally 30% of the cytosolic activity associated with membranes within 15 to 30 s, but most of this returned to the cytosol by 2 min. The small amount (3%) of PKC activity that remained associated with the membranes did so for at least 20 min but only if aggregation of the receptors was maintained. PKC translocation correlated with aggregation of receptors both at 30 s and at 10 min. However, only the translocation at 10 min and not that at 30 s correlated with receptor-induced exocytosis. In the absence of extracellular calcium (no exocytosis is observed), translocation at 30 s was diminished by 30% and at 10 min was completely absent. Cells depleted of PKC by 18-h treatment with PMA failed to degranulate in response to PMA and A23187 but responded partially (35%) when receptors were aggregated. We conclude that translocation of PKC is an early event that follows aggregation of IgE receptors but may not be essential for mediating the exocytotic mechanism induced by these receptors.     

Effects of epoxyeicosatrienoic acids on polymorphonuclear leukocyte function

During periods of ischemia and vascular injury, factors are released which recruit monocytes and polymorphonuclear leukocytes (PMNs) to the site of injury by promoting adherence to the endothelium and transmigration across the endothelial cell (EC) layer. During coronary artery stenosis, we have shown that the endothelium-derived, cytochrome P450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are elevated. Therefore, we examined if the EETs could stimulate PMN adherence to cultured ECs. Pretreatment of ECs with EETs for either 30 min or 4 hr did not alter the adherence of 51Cr-labelled PMNs to ECs while phorbol myristate acetate (PMA) produced a 4-fold increase in PMN adherence. The combination of EETs and PMA did not significantly augment or diminish PMA-induced PMN adherence to ECs. When ECs and 51Cr-labelled PMNs were coincubated, treatment with EETs alone did not alter PMN adherence. However, when EETs and PMA were added together during the coincubation of ECs and 51Cr-labelled PMNs, the EETs produced a concentration-related decrease in PMN adherence. Microscopic analysis of the culture media bathing the cells revealed aggregates of the labeled PMNs. We examined the effects of the EETs on PMN aggregation. 8,9-EET (10, 50, and 100 microM) increased PMN aggregation (7 +/- 3, 35 +/- 10, and 65 +/- 11%) and intracellular calcium by 1.7 +/- 0.5, 4.7 +/- 1.4, and 6.8 +/- 2.3-fold above basal. 5,6-, 11,2- and 14,15-EETs also stimulated aggregation. FMLP stimulated the production of superoxide; however, 8,9-EET did not. These observations indicate that the decrease in PMN adherence observed in the coincubation experiment is the result of EET-induced PMN aggregation. Given the increase in EET production during coronary artery stenosis, these data may provide insight into their potential biological significance during myocardial ischemia and vascular injury.     

Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils

In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.     

Opsonic properties of C-reactive protein. Stimulation by phorbol myristate acetate enables human neutrophils to phagocytize C-reactive protein-coated cells

We examined phagocytosis of sheep erythrocytes passively sensitized with pneumococcal C-polysaccharide (E-PnC) and of E-PnC coated with C-reactive protein (E-PnC-CRP) by human polymorphonuclear leukocytes (PMN). PMN isolated from blood of normal individuals failed to ingest either E-PnC or E-PnC-CRP; however, after stimulation with 12-O-tetradecanoylphorbol-13-acetate (PMA; 2 ng/ml), PMN ingested E-PnC-CRP efficiently with a mean phagocytic index (PI) of 99.5 +/- 4.8 (mean +/- SD, n = 11), and E-PnC to a lesser extent with a mean PI of 33.2 +/- 11.7 (mean +/- SD, n = 11). PMN that had adhered to PnC-coated glass and that were stimulated with PMA attached but did not ingest E-PnC-CRP. In contrast, PMN plated on E-PnC-CRP-coated glass and stimulated with PMA did not attach or ingest E-PnC-CRP. These data indicate that PMN can be induced to phagocytize PnC-CRP and that both PnC and CRP are required for ingestion. They also suggest that specific receptors for these ligands are expressed by stimulated PMN. Neither attachment nor phagocytosis of E coated with rabbit anti-E IgG (E-IgG) was affected by plating PMN on PnC or PnC-CRP. On the other hand, both phagocytosis and ingestion of E-PnC-CRP as well as E-IgG was blocked by plating PMA-stimulated PMN on immune complexes containing rabbit IgG. Inhibition experiments with the use of 3G8, a monoclonal antibody to the Fc gamma receptor of PMN, and human monomeric IgG1 demonstrated that attachment of E-PnC-CRP is mediated by receptors other than the Fc gamma receptors. These combined results indicated a nonreciprocal association between the putative CRP receptors and the Fc gamma receptors of stimulated PMN, resulting in the clearance of both types of receptors from the apical surface of PMN by antigen-immobilized rabbit IgG.     

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.     

The respiratory burst of bovine neutrophils. Role of a b type cytochrome and coenzyme specificity

A new method of preparation of bovine polymorphonuclear leukocytes (PMN) is described. The subcellular distribution of cytochrome b in resting and activated bovine PMN was compared to that of the O2-.-generating oxidase (assessed as NADPH cytochrome c reductase inhibited by superoxide dismutase). In resting PMN and in PMN activated by phorbol myristate acetate (PMA), cytochrome b was located into two membrane fractions, one of which was enriched in plasma membrane and cosedimented with alkaline phosphatase, while the other consisted of a denser material cosedimenting with markers of the specific and azurophil granules, i.e. the vitamin-B12-binding protein and myeloperoxidase respectively. During activation of PMN by PMA, 15-20% cytochrome b migrated from dense granules to the plasma membrane. The distribution of the O2-. generating oxidase and cytochrome b in subcellular particles was studied during the course of phagocytosis of PMA-coated latex beads by bovine PMN. At the onset of the respiratory burst, the phagocytic vacuoles arising from internalization of the plasma membrane were enriched in oxidase and alkaline phosphatase, but their specific content of cytochrome b was limited; in contrast, cytochrome b was predominant in denser membrane fractions cosedimenting with myeloperoxidase and the vitamin-B12-binding protein. After a few minutes of phagocytosis, a fraction of light vacuoles, slightly denser than the phagocytic vacuoles, became enriched in O2-.-generating oxidase, cytochrome b, the vitamin-B12-binding protein and myeloperoxidase. These vacuoles probably arose from the fusion of the phagocytic vacuoles with dense granules. In bovine PMN supplemented with glucose and maintained in anaerobiosis, activation by PMA induced slow reduction of cytochrome b (60-70% in 15 min at 37 degrees C). Similar results were obtained with cytoplasts after activation by PMA (30% reduction in 3 min at 37 degrees C). Cytochrome b in a particulate fraction obtained by centrifugation at 100 000 X g of an homogenate of PMA-activated PMN, was slowly reduced upon addition of NADPH under anaerobiosis (less 20% in 20 min at 37 degrees C). No reduction occurred in the 100 000 X g fraction prepared from non-activated PMN. The Soret band of cytochrome b reduced by dithionite was displaced by CO only by 1-2 nm. At subsaturating concentrations, CO had no effect on the rate of O2 uptake by activated bovine PMN.(ABSTRACT TRUNCATED AT 400 WORDS)