C-reactive protein mediates protection from lipopolysaccharide through interactions with Fc gamma R

C-reactive protein (CRP) is a component of the acute phase response to infection, inflammation, and trauma. A major activity of acute phase proteins is to limit the inflammatory response. It has been demonstrated that CRP protects mice from lethal doses of LPS. In the mouse, CRP binds to the regulatory receptor, FcgammaRIIb, and to the gamma-chain-associated receptor, FcgammaRI. The goal ofthis study was to determine whether FcgammaRs are necessary for the protective effect of CRP. The ability of CRP to protect mice from a lethal dose of LPS was confirmed using injections of 500 and 250 micro g of CRP at 0 and 12 h. CRP treatment of FcgammaRIIb-deficient mice increased mortality after LPS challenge and increased serum levels of TNF and IL-12 in response to LPS. CRP did not protect FcR gamma-chain-deficient mice from LPS-induced mortality. Treatment of normal mice, but not gamma-chain-deficient mice, with CRP increased IL-10 levels following LPS injection. In vitro, in the presence of LPS, CRP enhanced IL-10 synthesis and inhibited IL-12 synthesis by bone marrow macrophages from normal, but not gamma-chain-deficient mice. The protective effect of CRP appears to be mediated by binding to FcgammaRI and FcgammaRII resulting in enhanced secretion of the anti-inflammatory cytokine IL-10 and the down-regulation of IL-12. These results suggest that CRP can alter the cytokine profile of mouse macrophages by acting through FcgammaR leading to a down-regulation of the inflammatory response.     

Alteration of the Fc gamma RIIa dimer interface affects receptor signaling but not ligand binding

The aggregation of cell surface FcRs by immune complexes induces a number of important Ab-dependent effector functions. However, despite numerous studies that examine receptor function, very little is known about the molecular organization of these receptors within the cell. In this study, protein complementation, mutagenesis, and ligand binding analyses demonstrate that human FcgammaRIIa is present as a noncovalent dimer form. Protein complementation studies found that FcgammaRIIa molecules are closely associated. Mutagenesis of the dimer interface, as identified by crystallographic analyses, did not affect ligand binding yet caused significant alteration to the magnitude and kinetics of receptor phosphorylation. The data suggest that the ligand binding and the dimer interface are distinct regions within the receptor, and noncovalent dimerization of FcgammaRIIa may be an essential feature of the FcgammaRIIa signaling cascade.     

Serum amyloid P component and C-reactive protein mediate phagocytosis through murine Fc gamma Rs

The pentraxins, serum amyloid P component (SAP) and C-reactive protein (CRP) are acute-phase serum proteins in mice and humans, respectively. Although SAP binds to DNA and chromatin and affects clearance of these autoantigens, no specific receptor for SAP has been identified. CRP is an opsonin, and we have shown that it binds to FcgammaR. Mice deficient in FcgammaR were used to assess the role of these receptors in phagocytosis by pentraxins using zymosan as a ligand. Phagocytosis of zymosan by bone marrow macrophages (BMM) was enhanced by opsonization with SAP or CRP. BMM from mice deficient in all three FcgammaR or in gamma-chain ingested unopsonized zymosan, but phagocytosis of SAP- or CRP-opsonized zymosan was not enhanced. SAP binding to BMM from gamma-chain-deficient mice was also greatly reduced, indicating little or no binding of SAP to FcgammaRII. SAP and CRP opsonized zymosan for phagocytosis by BMM from mice deficient in FcgammaRII or FcgammaRIII. SAP, but not CRP, opsonized zymosan for uptake by neutrophils that express only low levels of FcgammaRI. Together these results indicate that FcgammaRI and FcgammaRIII are receptors for SAP in the mouse. Opsonization of zymosan by CRP is mediated through FcgammaRI. Pentraxins are major proteins of the innate immune system and arose earlier in evolution than Igs. The use of FcgammaR by the pentraxins links innate and adaptive immunity and may have important consequences for processing, presentation, and clearance of the self-Ags to which these proteins bind.     

C-reactive protein increases cytokine responses to Streptococcus pneumoniae through interactions with Fc gamma receptors

Streptococcus pneumoniae is the most common organism responsible for community acquired pneumonia and meningitis. In pneumococcal pneumonia, a strong local inflammatory cytokine response reduces the frequency of bacteremia and increases survival. The initiation of this cytokine response by innate recognition of bacterial cell wall components through TLR has been described, but the role of soluble innate mediators has received limited attention. C-reactive protein (CRP) is an acute phase protein that binds phosphocholine residues on S. pneumoniae cell walls. CRP interacts with phagocytic cells through FcgammaRI and FcgammaRII and activates the classical complement pathway. CRP is protective in mouse pneumococcal bacteremia by increasing complement-dependent clearance and killing of bacteria. We studied the cytokine response of PBMC stimulated with CRP-opsonized S. pneumoniae to determine the effect of CRP interaction with FcgammaR. CRP dramatically increased the production of TNF-alpha and IL-1beta in response to S. pneumoniae. These increases were blocked by phosphocholine, which inhibits CRP binding to S. pneumoniae, by inhibitors of FcgammaR signaling, and by mAb to FcgammaRI and FcgammaRII. A mutated rCRP with decreased FcgammaR binding had a decreased ability to stimulate TNF-alpha release, compared with wild-type CRP. Individuals who were homozygous for the R-131 allele of FcgammaRIIA, which has a higher affinity for CRP, showed higher responses to CRP-opsonized bacteria than did individuals homozygous for the H-131 allele, further implicating this receptor. The results indicate that CRP recognition of S. pneumoniae and binding to FcgammaR may enhance the early protective cytokine response to infection.     

Fc gamma RIIa, not Fc gamma RIIb, is constitutively and functionally expressed on skin-derived human mast cells

The expression of FcgammaR by human skin-derived mast cells of the MC(TC) type was determined in the current study. Expression of mRNA was analyzed with microarray gene chips and RT-PCR; protein by Western blotting and flow cytometry; function by release of beta-hexosaminidase, PGD(2), leukotriene C(4) (LTC(4)), IL-5, IL-6, IL-13, GM-CSF, and TNF-alpha. FcgammaRIIa was consistently detected along with FcepsilonRI at the mRNA and protein levels; FcgammaRIIc was sometimes detected only by RT-PCR; but FcgammaRIIb, FcgammaRI, and FcgammaRIII mRNA and protein were not detected. FcgammaRIIa-specific mAb caused skin MC(TC) cells to degranulate and secrete PGD(2), LTC(4), GM-CSF, IL-5, IL-6, IL-13, and TNF-alpha in a dose-dependent fashion. FcepsilonRI-specific mAb caused similar amounts of each mediator to be released with the exception of LTC(4), which was not released by this agonist. Simultaneous but independent cross-linking of FcepsilonRI and FcgammaRIIa did not substantially alter mediator release above or below levels observed with each agent alone. Skin MC(TC) cells sensitized with dust-mite-specific IgE and IgG, when coaggregated by Der p2, exhibited enhanced degranulation compared with sensitization with either IgE or IgG alone. These results extend the known capabilities of human skin mast cells to respond to IgG as well as IgE-mediated signals.     

C-reactive protein binding to murine leukocytes requires Fc gamma receptors

Human C-reactive protein (CRP) is an acute phase protein that binds to receptors on human and mouse leukocytes. We have recently determined that the high and low affinity receptors for CRP on human leukocytes are Fc gamma RIIa and Fc gamma RI, respectively. Previous work by others suggested that CRP receptors on mouse macrophages are distinct from Fc gamma R. We have taken advantage of the availability of mice deficient in one or more Fc gamma R to reexamine the role of Fc gamma R in CRP binding to mouse leukocytes. Three strains of Fc gamma R-deficient mice were examined: gamma-chain-deficient mice that lack Fc gamma RI and Fc gamma RIII, Fc gamma RII-deficient mice, and mice deficient in both gamma-chain and Fc gamma RII that lack all Fc gamma R. No binding of CRP was detected to leukocytes from double-deficient mice, indicating that Fc gamma R are required for CRP binding. CRP binding to leukocytes from gamma-chain-deficient and Fc gamma RII-deficient mice was reduced compared with binding to leukocytes from wild-type mice. Further analysis of CRP binding to macrophages, neutrophils, and lymphocytes provides direct evidence that Fc gamma RIIb1, Fc gamma RIIb2, and Fc gamma RI are the receptors for CRP on mouse leukocytes. These findings may have important implications in understanding the physiological function of CRP.     

Decreased expression of Fc gamma RIII mRNA in leukemic granulocytes.

Morphologically mature granulocytes from patients with chronic myeloid leukemia show significant impairment in their ability to internalize aggregated IgG, a ligand that is rapidly phagocytosed by normal human granulocytes. With a view to understand the molecular basis of this defect, normal and leukemic granulocytes were examined for the steady-state levels of mRNA for Fc gamma RIII, a membrane-associated receptor that initially binds and traps the IgG-opsonized antigens. Northern blot analyses revealed that the level of the specific mRNA in CML granulocytes was between 0.08 and 0.69 times that seen in the normal granulocytes. This could be one of the contributory factors for the observed endocytic defect in the leukemic granulocytes.     

Geranylgeranylacetone induces apoptosis in HL-60 cells.

Geranylgeranylacetone (GGA) induces apoptosis in human leukemia HL-60 cells in a dose- and time-dependent manner. This effect was completely prevented by the pan-caspase inhibitor z-Val-Ala-Asp(OMe) fluoromethylketone, thereby implicating the caspase cascade in the process. Prior to DNA fragmentation, GGA treatment markedly activated caspase-3(-like) proteases, which might be responsible for the observed apoptosis. In addition, GGA treatment interfered with the processing and membrane localization of Rap1 and Ras, and these changes may be a result of apoptosis. Moreover, nitric oxide donors significantly accentuated the GGA-induced apoptosis, suggesting that the apoptotic pathway induced by GGA might be regulated by a redox-sensitive mechanism. Taken together, these data suggest that the isoprenoid, GGA, is an effective inducer of apoptotic cell death in HL-60 cells.     

Activation of Fc gamma RII induces tyrosine phosphorylation of multiple proteins including Fc gamma RII.

Platelets provide a useful system for studying Fc gamma receptor-mediated signaling events because these cells express only a single class of Fc gamma receptors and because platelet aggregation and secretion can be activated through Fc gamma receptor stimulation. We report here that stimulation of platelets by cross-linking antibodies to Fc gamma RII or by treatment with an anti-CD9 monoclonal antibody, which acts through Fc gamma RII, causes an induction of tyrosine phosphorylation of multiple platelet proteins. Although the profile of tyrosine-phosphorylated proteins induced by stimulation of this Fc receptor was similar to that induced by thrombin, an additional 40-kDa phosphorylated protein was also detected. This protein co-migrated with Fc gamma RII and was immunoprecipitated with a monoclonal antibody to Fc gamma RII. In addition, after the cross-linking of Fc gamma RII in HEL cells or in COS-1 cells transfected with Fc gamma RII cDNA, the 40-kDa protein immunoprecipitated with anti-Fc gamma RII was also phosphorylated on tyrosine. These data strongly suggest that Fc gamma RII itself is a substrate for a tyrosine kinase(s) activated when Fc gamma RII is stimulated. Fc gamma RII was phosphorylated by the Src protein in vitro, suggesting that this kinase may be responsible for phosphorylation of Fc gamma RII in vivo. These studies establish that activation of platelets and human erythroleukemia cells through Fc gamma RII and CD9 involves an induction of tyrosine phosphorylation of multiple proteins including Fc gamma RII itself and suggest that these phosphorylation events may be involved in Fc gamma RII-mediated cell signaling.     

Signal transduction by human-restricted Fc gamma RIIa involves three distinct cytoplasmic kinase families leading to phagocytosis.

Recent experiments indicate an important role for Src family and Syk protein tyrosine kinases and phosphatidylinositol 3-kinase in the signal transduction process initiated by mouse receptors for IgG and leading to phagocytosis. Considerably less is known regarding signal transduction by the human-restricted IgG receptor, FcgammaRIIa. Furthermore, the relationship among the Src family, Syk, and phosphatidylinositol 3-kinase in phagocytosis is not understood. Here, we show that FcgammaRIIa is phosphorylated by an Src family member, which results in recruitment and concomitant activation of the distal enzymes Syk and phosphatidylinositol 3-kinase. Using a FcgammaRI-p85 receptor chimera cotransfected with kinase-inactive mutants of Syk or application of a pharmacological inhibitor of Syk, we show that Syk acts in parallel with phosphatidylinositol 3-kinase. Our results indicate that FcgammaRIIa-initiated monocyte or neutrophil phagocytosis proceeds from the clustered IgG receptor to Src to phosphatidylinositol 3-kinase and Syk.     

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.     

Platelet activation induces increased Fc gamma receptor expression

Platelet contain Fc gamma RII. However, little is known about how the expression of these receptors is regulated. Inasmuch as platelet activation by a variety of agonists increases the expression of several proteins on the platelet surface, we used flow cytometry to study the effect of platelet activation on the expression of platelet Fc gamma R by measuring the binding of fluorescein-labeled oligomeric IgG (FITC-IgG oligomer) and fluorescein-labeled mAb IV.3 (FITC-IV.3), a mAb that recognizes Fc gamma RII, to platelets. The number of Fc gamma R per platelet was determined by relating the binding to platelets of FITC-IV.3, measured by flow cytometry, to the binding of 125I-labeled IV.3, measured using a standard filtration assay. Nonactivated, gel-filtered platelets from nine healthy donors expressed a mean of 891 Fc gamma R per platelet, whereas platelets activated at 25 degrees C by thrombin or PMA expressed a mean of 1382 Fc gamma R an average increase of 55% (p less than 0.001). Binding of FITC-IgG oligomer increased to a similar extent when platelets were stimulated by these agonists. A smaller increase in the number of Fc gamma R expressed on the platelet surface was measured when platelets were stimulated with ADP, though no increase was observed with epinephrine. The agonist-dependent increase in Fc gamma R expression did not occur when platelets were studied at 4 degrees C or in the presence of agents that elevate intracellular levels of cAMP, suggesting that platelet activation was required for this process. Agonist-stimulated Fc gamma R expression did not depend on dense-granule secretion, because it was observed at low agonist concentrations in the absence of 14C-serotonin release. These studies demonstrate that the number of Fc gamma R expressed on the platelet surface increases when platelets are activated by several agonists, perhaps as a result of the exposure of Fc gamma R located along the surface-connected open canalicular system, or the fusion of platelet alpha-granule and plasma membranes during the activation process. Increased Fc gamma R expression may promote the clearance of IgG-containing immune complexes from the circulation, and contribute to the development of immune complex-mediated thrombocytopenia.     

C-reactive protein receptors on the human monocytic cell line U-937. Evidence for additional binding to Fc gamma RI.

C-reactive protein (CRP) is an acute-phase protein that binds to components of damage tissue, activates C, and stimulates phagocytic cells. CRP binding to receptors on monocytic and polymorphonuclear phagocytes has been shown. Recently, CRP-binding proteins of 38 to 40 kDa and 57 to 60 kDa have been identified on the human promonocyte cell line U-937 and the mouse macrophage cell line PU5 1.8, respectively. However, analysis of CRP binding to these cells and to peripheral blood leukocytes suggests that additional CRP receptor sites may be present. Because many studies have shown interactions between CRP binding and IgG binding to leukocytes, we have examined further the CRP binding sites on U-937 cells and determined their relationship to the FcR for IgG (Fc gamma R) expressed on these cells. Our results demonstrate specific saturable binding of CRP to peripheral blood monocytes and U-937 cells, which is readily inhibited by aggregated IgG. Monomeric IgG, which binds specifically to Fc gamma RI, inhibited a maximum of 20% of CRP binding to these cells. mAb 197 and mAb IV.3, which block IgG binding to Fc gamma RI and Fc gamma RII, respectively, failed to inhibit CRP binding to U-937 cells. Two CRP-binding molecules were identified by precipitation of lysates from surface-labeled U-937 cells and cross-linking experiments. One of these had a molecular mass of 43 to 45 kDa, similar to the molecule previously described as the CRPR on U-937 cells. The other had the same mobility by SDS-PAGE as Fc gamma RI. The identity of this protein with Fc gamma RI was confirmed by the ability of both IgG-Sepharose and CRP-Sepharose to preclear the protein from cell lysates and by inhibition of binding to both IgG-Sepharose and CRP-Sepharose by anti-Fc gamma RI mAb 197.     

Convergence of Fc gamma receptor IIA and Fc gamma receptor IIIB signaling pathways in human neutrophils

Human neutrophils (PMNs) express two receptors for the Fc domain of IgG: the transmembrane FcgammaRIIA, whose cytosolic sequence contains an immunoreceptor tyrosine-based activation motif, and the GPI-anchored FcgammaRIIIB. Cross-linking of FcgammaRIIIB induces cell activation, but the mechanism is still uncertain. We have used mAbs to cross-link selectively each of the two receptors and to assess their signaling phenotypes and functional relation. Cross-linking of FcgammaRIIIB induces intracellular Ca2+ release and receptor capping. The Ca2+ response is blocked by wortmannin and by N,N-dimethylsphingosine, inhibitors of phosphatidylinositol 3-kinase and sphingosine kinase, respectively. Identical dose-response curves are obtained for the Ca2+ release stimulated by cross-linking FcgammaRIIA, implicating these two enzymes in a common signaling pathway. Wortmannin also inhibits capping of both receptors, but not receptor endocytosis. Fluorescence microscopy in double-labeled PMNs demonstrates that FcgammaRIIA colocalizes with cross-linked FcgammaRIIIB. The signaling phenotypes of the two receptors diverge only under frustrated phagocytosis conditions, where FcgammaRIIIB bound to substrate-immobilized Ab does not elicit cell spreading. We propose that FcgammaRIIIB signaling is conducted by molecules of FcgammaRIIA that are recruited to protein/lipid domains induced by clustered FcgammaRIIIB and, thus, are brought into juxtaposition for immunoreceptor tyrosine-based activation motif phosphorylation and activation of PMNs.     

Fc gamma receptor III induces actin polymerization in human neutrophils and primes phagocytosis mediated by Fc gamma receptor II.

Human polymorphonuclear leukocytes (PMN) express two classes of Fc gamma R: Fc gamma RII the 42-kDa receptor with a traditional membrane spanning domain and cytoplasmic tail and Fc gamma RIIIPMN the 50- to 80-kDa receptor with a glycosyl-phatidylinositol membrane anchor expressed on PMN. To explore the capacity of Fc gamma RIIIPMN to generate intracellular signals, we have analyzed the ability of Fab and F(ab')2 anti-Fc gamma R mAb to induce actin filament assembly, a prerequisite for motile behaviors. Multivalent ligation of Fc gamma RIIIPMN, independent of Fc gamma RII, results in an increase in F-actin content that is [Ca2+]i dependent. Multivalent ligation of Fc gamma RII also initiates actin polymerization but uses a [Ca2+]i-independent initial pathway. In addition to providing a mechanism for Fc gamma RIIIPMN triggered effector functions, the increase in F-actin and [Ca2+]i generated by Fc gamma RIIIPMN ligation also serves as a "priming" signal to modify PMN responses to other stimuli. Experiments using erythrocytes specifically coated with anti-Fc gamma RII Fab demonstrate that cross-linking of Fc gamma RIIIPMN with anti-Fc gamma RIII F(ab')2 enhances phagocytosis mediated by Fc gamma RII. Thus, Fc gamma RIIIPMN, a glycosyl-phosphatidylinositol anchored protein, may contribute directly to an intracellular program of actin assembly that may trigger and prime neutrophil effector functions.     

Phospholipase D in homogenates from HL-60 granulocytes: implications of calcium and G protein control

Occupancy of chemotactic peptide receptors leads to rapid initiation of phospholipase D (PLD) activity in intact dimethylsulfoxide-differentiated HL-60 granulocytes (Pai, J.-K, Siegel, M.I., Egan, R.W., and Billah, M.M. (1988) J. Biol. Chem. 263, 12472). To gain further insight into the activation mechanisms, PLD has been studied in cell lysates from HL-60 granulocytes, using 1-0-alkyl-2-oleoyl-[32P]phosphatidylcholine (alkyl-[32P]PC), 1-0-[3H]alkyl-2-oleoyl-phosphatidylcholine [( 3H]alkyl-PC) and [14C]arachidonyl-phospholipids as substrates. In the presence of Ca2+ and GTP gamma S, post-nuclear homogenates degrade alkyl-[32P]PC to produce 1-0-alkyl-[32P]phosphatidic acid (alkyl-[32P]-PA), and in the presence of ethanol, also 1-0-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). By comparing the 3H/32P ratios of PA and PEt to that of PC, it is concluded that PA and PEt are formed exclusively by a PLD that catalyzes both hydrolysis and transphosphatidylation between PC and ethanol. Furthermore, PC containing either ester- or ether-linkage at the sn-1 position is degraded in preference to phosphatidylethanolamine and phosphatidylinositol by PLD in HL-60 cell homogenates. It is concluded that HL-60 granulocytes contain a PC-specific PLD that requires both Ca2+ and GTP for activation.     

Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells

Most cell lines that lack functional p53 protein are arrested in the G₂ phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G₂ phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G₂ phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D₀ value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G₂ phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D₀=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5–7post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.     

C-reactive protein enhances immunity to Streptococcus pneumoniae by targeting uptake to Fc gamma R on dendritic cells

C-reactive protein (CRP) is an acute phase reactant with roles in innate host defense, clearance of damaged cells, and regulation of the inflammatory response. These activities of CRP depend on ligand recognition, complement activation, and binding to FcgammaR. CRP binds to phosphocholine in the Streptococcus pneumoniae cell wall and provides innate defense against pneumococcal infection. These studies examine the effect of this early innate defense molecule on the development of Abs and protective immunity to S. pneumoniae. Dendritic cells (DC) initiate and direct the adaptive immune response by integrating innate stimuli with cytokine synthesis and Ag presentation. We hypothesized that CRP would direct uptake of S. pneumoniae to FcgammaR on DC and enhance Ag presentation. CRP opsonization of the R36a strain of S. pneumoniae increased the uptake of bacteria by DC. DC pulsed with untreated or CRP-opsonized R36a were transferred into recipient mice, and Ab responses were measured. In mice challenged with free R36a, CRP opsonization resulted in higher secondary and memory IgG responses to both phosphocholine and pneumococcal surface protein A. Furthermore, mice immunized with DC that had been pulsed with CRP-opsonized R36a showed increased resistance to intranasal infection with virulent S. pneumoniae. The effects of CRP on Ag uptake, Ab responses, and protection from infection all required FcR gamma-chain expression on DC. The results indicate that innate recognition by CRP enhances effective uptake and presentation of bacterial Ags through FcgammaR on DC and stimulates protective adaptive immunity.     

Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes

There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-[32P] lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-[32P]PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-[32P]PA must be formed via PLD-catalyzed hydrolysis of alkyl-[32P]PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Formation of alkyl-[32P]PEt parallels that of alkyl-[32P]PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration. These results strongly support the hypothesis that in HL-60 granulocytes, PEt is formed via PLD-catalyzed transphosphatidylation. Moreover, using HL-60 granulocytes double-labeled by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC, it has been established that the early (30 s) appearance of alkyl-PA is due primarily to PLD, not phospholipase C as previously thought, and that alkyl-PEt is formed exclusively by PLD. These results constitute the first direct evidence for receptor-linked activation of PLD, leading to the generation of PA and PEt in an intact cell system.     

Activation of phospholipase D by chemotactic peptide in HL-60 granulocytes

Activation of phospholipase D (PLD) has been investigated in dimethylsulfoxide differentiated HL-60 granulocytes labeled in endogenous 1-0-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) by incubation with [3H]alkyl-lysoPC. Stimulation of these labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), induces rapid generation of [3H]phosphatidic acid (PA) and slower formation of [3H]diglyceride, suggesting hydrolysis of alkyl-PC by PLD. A unique feature of PLD is its ability to transfer the phosphatidyl moiety of phospholipids to alcohols (transphosphatidylation). This characteristic has been exploited to identify PLD activity. For example, when ethanol is present during stimulation of the HL-60 cells, [3H]phosphatidylethanol (PEt) is formed with a concomitant decrease in [3H]PA. Cells incubated with [32P]orthophosphate to label the terminal phosphate of ATP do not incorporate 32P into PEt, consistent with the [3H]PEt not being synthesized from [3H]diglyceride. In contrast, [3H]PA arises from both PLD and diglyceride kinase activities. Furthermore, PEt synthesis closely parallels PA formation and both are inhibited by an fMLP receptor antagonist, suggesting that both PA and PEt are derived from agonist-stimulated PLD action. These observations are consistent with phospholipase D-catalyzed breakdown of alkyl-PC in fMLP- stimulated granulocytes.