Natural killing and antibody-dependent cellular cytotoxicity are mediated by different mechanisms and by different cells.

Natural killing (NK) in humans, as well as in other species, has been shown to be specific for antigenic determinants present on the surfaces of a variety of tumor cells. Physical separation of NK cells from K cells, which mediate antibody-dependent cellular cytotoxicity (ADCC), has not been successful; however, there is indirect evidence suggesting that these activities are distinct. To further study the relationship between NK and K cells, competitive inhibition techniques were employed. NK cells can be blocked via two mechanisms: 1) by direct inhibition with NK-sensitive tumor cells binding to NK receptor sites present on the effector cells and 2) by steric inhibition resulting from the binding of antibody-coated cells to the FcR on the effector cells. K cells, however, lack the NK receptor site(s) but are FcR+, and can therefore be blocked only by antibody-coated cells. We therefore postulate that NK and K cells are two separate lymphoid populations. NK cells bear receptor site(s) for NK determinants and FcR, whereas K cells bear only FcR.     

Platelets and parotid acinar cells have different mechanisms for agonist-stimulated divalent cation entry

Stimulation of platelets with thrombin or parotid acinar cells with carbachol results in an increase in [Ca2+]i which is due to both release from internal stores and influx across the plasma membrane. In platelets, thrombin also stimulates Mn2+ entry into the cytosol; this is seen as a stimulated quench of fura-2 fluorescence. In the parotid, however, carbachol does not stimulate Mn2+ entry. This result suggests different mechanisms of stimulated divalent cation entry in the two cell types and could have important implications in the study of receptor-mediated Ca2+ entry mechanisms.     

Plasmacytoid dendritic cells of different origins have distinct characteristics and function: studies of lymphoid progenitors versus myeloid progenitors

Plasmacytoid dendritic cells (pDCs) play a central role in host innate and adaptive immunity and are thought to be of lymphoid origin. However, in IL-7Ralpha-/- mice, which are deficient in T and B lymphocytes, pDCs are still found in lymphoid organs, which suggests that there is a lymphoid-independent pathway for the development of pDCs. Previous work has demonstrated that pDCs originate from both lymphoid and myeloid progenitors (MPs). However, it is not clear whether the function of pDCs is different relative to their origin. In an effort to compare the characteristics and functions between pDCs generated from different progenitors, we performed adoptive transfer studies using highly enriched populations of common lymphoid progenitors (CLPs) and MPs from the bone marrow of control mice and examined their potential and developmental kinetics for the generation of pDCs. Interestingly, although CLPs were polarized to generate pDCs, MPs were polarized to generate conventional dendritic cells and the kinetics of pDC generation from MPs was reached earlier than from CLPs. Furthermore, CLPs have the potential to generate more pDCs on a per cell basis. Moreover, MP-derived pDCs produce relatively higher levels of IFN-alpha than CLP-derived pDCs following CpG stimulation. These data indicate that MPs are multipotential and have the capacity to develop into not only myeloid cells, but also pDCs, which have distinct characteristics and function compared to that of lymphoid origin and, therefore, imply a more important role for MP-derived pDCs in conditions where the function of lymphoid progenitors is impaired or compromised.     

Purinergic receptor-induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium-related mechanisms

In human cervical (CaSki) cells, extracellular adenosine triphosphate (ATP) induces an acute decrease in the resistance of the lateral intercellular space (R _LIS), phase I response, followed by an increase in tight junctional resistance (R _TJ), phase II response. ATP also stimulates release of calcium from intracellular stores, followed by augmented calcium influx, and both effects have similar sensitivities to ATP (EC₅₀ of 6 μM). The objective of the study was to determine the degree to which the changes in [Ca²⁺]_i mediate the responses to ATP. 1,2-bis (2-aminophenoxy) ethane-N,N,N1,N1-tetraacetic acid (BAPTA) abrogated calcium mobilization and phase I response; in contrast, nifedipine and verapamil inhibited calcium influx and attenuated phase II response. Barium, La³⁺, and Mn²⁺ attenuated phase I response and attenuated and shortened the ionomycin-induced phase I-like decrease inR _LIS, suggesting that store depletion-activated calcium entry was inhibited. Barium and La³⁺ also inhibited the ATP-induced phase II response, but Mn²⁺ had no effect on phase II response, and in the presence of low extracellular calcium it partly restored the increase inR _TJ. KCl-induced membrane depolarization stimulated an acute decrease inR _LIS and a late increase inR _TJ similar to ATP, but only the latter was inhibited by nifedipine. KCl also induced a nifedipine-sensitive calcium influx, suggesting that acute increases in [Ca²⁺]_i, regardless of mobilization or influx, mediate phase I response. Phase II-like increases inR _TJ could be induced by treatment with diC8, and were not affected by nifedipine. Biphasic ATP-like changes inR _TE could be induced by treating the cells with ionomycin plus diC8. We conclude that calcium mobilization mediates the early decrease inR _LIS, and calcium influx via calcium channels activates protein kinase C and mediates the late increase inR _TJ.     

The different morphologies of yeast and filamentous fungi trigger distinct killing and feeding mechanisms in a fungivorous amoeba

Size and diverse morphologies pose a primary challenge for phagocytes such as innate immune cells and predatory amoebae when encountering fungal prey. Although filamentous fungi can escape phagocytic killing by pure physical constraints, unicellular spores and yeasts can mask molecular surface patterns or arrest phagocytic processing. Here, we show that the fungivorous amoeba Protostelium aurantium was able to adjust its killing and feeding mechanisms to these different cell shapes. Yeast-like fungi from the major fungal groups of basidiomycetes and ascomycetes were readily internalized by phagocytosis, except for the human pathogen Candida albicans whose mannoprotein coat was essential to escape recognition by the amoeba. Dormant spores of the filamentous fungus Aspergillus fumigatus also remained unrecognized, but swelling and the onset of germination induced internalization and intracellular killing by the amoeba. Mature hyphae of A. fumigatus were mostly attacked from the hyphal tip and killed by an actin-mediated invasion of fungal filaments. Our results demonstrate that predatory pressure imposed by amoebae in natural environments selects for distinct survival strategies in yeast and filamentous fungi but commonly targets the fungal cell wall as a crucial molecular pattern associated to prey and pathogens.     

The effect of monensin on the antibody-dependent cell-mediated killing

Antibody-dependent cell-mediated immune cytolysis of herpes simplex virus-infected target cells involves two separate events, recognition and adhesion of the effector K cells to the antibody-coated target cells, and the final killing step. In the present study the killing event is shown to be dependent on an intact secretory mechanism in the K cells. Treatment of K cells with the carboxylic ionophore monensin, which blocks secretion, completely abolishes the K-cell-mediated killing, but the adhesion of the effector cells to the antibody-coated target cells is not affected by monensin. The similarity between the killing events mediated by K and NK cells is discussed.     

EGF AND TGF-alpha motogenic activities are mediated by the EGF receptor via distinct matrix-dependent mechanisms

EGF and TGF-alpha induce an equipotent stimulation of fibroblast migration and proliferation. In spite of their homologous structure and ligation by the same receptor (EGFR), we report that their respective motogenic activities are mediated by different signal transduction intermediates, with p70(S6K) participating in EGF signalling and phospholipase Cgamma in TGF-alpha signalling. We additionally demonstrate that EGF and TGF-alpha motogenic activities may be resolved into two stages: (a) cell "activation" by a transient exposure to either cytokine, and (b) the subsequent "manifestation" of an enhanced migratory phenotype in the absence of cytokine. The cell activation and manifestation stages for each cytokine are mediated by distinct matrix-dependent mechanisms: motogenetic activation by EGF requires the concomitant functionality of EGFR and the hyaluronan receptor CD44, whereas activation by TGF-alpha requires EGFR and integrin alphavbeta3. Manifestation of elevated migration no longer requires the continued presence of exogenous cytokine and functional EGFR but does require the above mentioned matrix receptors, as well as their respective ligands, i.e., hyaluronan in the case of EGF, and vitronectin in the case of TGF-alpha. In contrast, the mitogenic activities of EGF and TGF-alpha are independent of CD44 and alphavbeta3 functionality. These results demonstrate clear qualitative differences between EGF and TGF-alpha pathways and highlight the importance of the extracellular matrix in regulating cytokine bioactivity.     

Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition are mediated by the packing parameter of phospholipid-detergent systems

The detergent solubilization and reformation of phospholipid vesicles was studied for various detergents. Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition were observed by turbidimetry and cryo-electron microscopy. The first mechanism involves fast solubilization of phospholipids and occurs via open vesicular intermediates. The reverse process, micelle-to-vesicle transition, mimics the vesicle-to-micelle transition. In the second mechanism the solubilization is a slow process that proceeds via micelles that pinch off from closed vesicles. During vesicle reformation, the micelle-to-vesicle transition, a large number of densely packed multilamellar vesicles are formed. The route used, for solubilization and reformation, by a given detergent-phospholipid combination is critically dependent on the overall packing parameter of the detergent-saturated phospholipid membranes. By a change of the overall packing parameter the solubilization and or reformation mechanism could be changed. All five detergents tested fit within the proposed model. With two detergents the mechanism could be changed by changing the phospholipid composition or the medium conditions.     

Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition are mediated by the packing parameter of phospholipid-detergent systems

The detergent solubilization and reformation of phospholipid vesicles was studied for various detergents. Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition were observed by turbidimetry and cryo-electron microscopy. The first mechanism involves fast solubilization of phospholipids and occurs via open vesicular intermediates. The reverse process, micelle-to-vesicle transition, mimics the vesicle-to-micelle transition. In the second mechanism the solubilization is a slow process that proceeds via micelles that pinch off from closed vesicles. During vesicle reformation, the micelle-to-vesicle transition, a large number of densely packed multilamellar vesicles are formed. The route used, for solubilization and reformation, by a given detergent-phospholipid combination is critically dependent on the overall packing parameter of the detergent-saturated phospholipid membranes. By a change of the overall packing parameter the solubilization and or reformation mechanism could be changed. All five detergents tested fit within the proposed model. With two detergents the mechanism could be changed by changing the phospholipid composition or the medium conditions.     

The hepatic galactosyl receptor system: two different ligand dissociation pathways are mediated by distinct receptor populations

After internalization of 125I-asialo-orosomucoid (ASOR) by isolated rat hepatocytes, ligand dissociates by two kinetically distinct pathways (Oka and Weigel, J. Biol. Chem. 257, 10,253, 1983). These slow and fast dissociation pathways correspond to two functionally different subpopulations of cell surface galactosyl receptors designated, respectively, State 1 and State 2 receptors. Freshly isolated cells or cells equilibrated below 24 degrees C express only State 1 receptors. Cells equilibrated at 37 degrees C express both State 1 and State 2 receptors. Ligand dissociation after internalization of surface-bound 125I-ASOR was measured using the permeabilizing detergent, digitonin. The slow dissociation pathway was mediated by State 1 receptors and was the only pathway expressed by cells which were freshly isolated or had been equilibrated at 24 degrees C. State 2 receptors are expressed at temperatures above about 20 degrees C, and both the fast and slow dissociation pathways occurred in cells equilibrated at 37 degrees C. State 2 receptors therefore mediate the rapid dissociation pathway. Dissociation and subsequent degradation of specifically bound ligand routed in either pathway were complete, respectively, within 3 and 6 hrs.     

Trypanosoma cruzi: antibody-dependent killing of bloodstream trypomastigotes by mouse bone marrow-derived mast cells and by mastocytoma cells

Two different populations of mast cells, that is, mastocytoma cells (P815) that were maintained either in vitro or in vivo, and mast cells obtained by differentiation of bone marrow precursor cells (MMC) in conditioned medium, were used as effector cells in antibody-dependent cytotoxic reactions (ADCC) against bloodstream trypomastigotes (BT) of Trypanosoma cruzi. The assay consisted of incubating effector cells with parasites that had been previously sensitized with immune mouse sera, immune IgG isotypes, or with medium. After the incubation period, the number of live BT was assessed. It was found that (a) cytotoxicity is antibody dependent; (b) the main isotypes involved are IgG1, IgG2a, and IgG2b; (c) both types of mast cells (mastocytoma and MMC cells) are equally efficient in killing BT; (d) mastocytoma cells degranulated by pretreatment with compound 48/80 are still able to effect ADCC; (e) on optical microscope examination, large numbers of parasites were often seen attached to the cells, but only when anti-T. cruzi antibodies were present; and (f) on electron microscope examination, no integral or ruptured parasites were seen inside the cells. We conclude that both T dependent and T independent mast cells are capable of mediating ADCC by a mechanism that is probably not dependent on granule extrusion.     

A comparison of antibody-dependent cellular cytotoxicity (ADCC) mediated by murine and human lymphoid cell populations

Mouse lymphoid cells are known to lyse chicken red blood cells (CRBC) in the presence of antibody and in the absence of complement. They have also been reported to effect lysis of mouse tumor cells and other nucleated targets, although this has been disputed. Using a 4-hr ⁵¹Cr-release assay, we have compared the activity of mouse effector cells from the thymus, spleen, bone marrow, peritoneal cavity, and mesenteric and subcutaneous lymph nodes of many strains of mice to the activity of human lymphoid cell effectors against CRBC and a number of murine targets. Human effectors mediate lysis of all targets tested. Mouse effectors lyse CRBC, but usually less well than human effectors. Mouse cells from lymphoid organs were either very inefficient or completely inactive against nucleated mammalian targets under a range of test conditions. Interestingly, in experiments where cells from solid lymphoid organs or the peritoneal cavity were ineffective, peripheral blood lymphocytes from one subline of DBA/2 consistently gave significant lysis of EL4 targets, while cells from another subline of the same strain did not.     

Stress fibers are generated by two distinct actin assembly mechanisms in motile cells

Stress fibers play a central role in adhesion, motility, and morphogenesis of eukaryotic cells, but the mechanism of how these and other contractile actomyosin structures are generated is not known. By analyzing stress fiber assembly pathways using live cell microscopy, we revealed that these structures are generated by two distinct mechanisms. Dorsal stress fibers, which are connected to the substrate via a focal adhesion at one end, are assembled through formin (mDia1/DRF1)-driven actin polymerization at focal adhesions. In contrast, transverse arcs, which are not directly anchored to substrate, are generated by endwise annealing of myosin bundles and Arp2/3-nucleated actin bundles at the lamella. Remarkably, dorsal stress fibers and transverse arcs can be converted to ventral stress fibers anchored to focal adhesions at both ends. Fluorescence recovery after photobleaching analysis revealed that actin filament cross-linking in stress fibers is highly dynamic, suggesting that the rapid association-dissociation kinetics of cross-linkers may be essential for the formation and contractility of stress fibers. Based on these data, we propose a general model for assembly and maintenance of contractile actin structures in cells.     

Natural killing and antibody-dependent cellular cytotoxicity: characterization of effector cells by E-rosetting and monoclonal antibodies

Recent investigations have indicated that the OKM1 hybridoma monoclonal antibody reactive with cells of the myelomonocytic series identifies a subpopulation of human peripheral blood mononuclear cells (PBMNC) which mediate natural and antibody-dependent cellular cytotoxicity (ADCC). However, it was not clear whether this OKM1+ group was heterogeneous with regard to cytotoxic function or the presence of receptors for sheep erythrocytes. Thus, the purpose of the present study was to further define the phenotype of the ADCC effector cell and natural killer (NK) cell using a combination of reactivity with hybridoma antibodies and separation of subsets by sheep erythrocyte rosette (E+) formation. Furthermore, the phenotypes of the NK population were assessed directly by performing two-color immunofluorescent staining on tumor cell conjugates. These studies led to the following conclusions: (1) that NK activity is mediated by both E+ OKM1+ and E- OKM1+ cells; the E+ OKT3+ cell possessed essentially no ADCC or NK activity; (2) that E+ OKM1+ cells mediated more NK activity on a per cell basis than E- OKM1+ cells; this was verified by separating OKM1+ cells on a cell sorter into E+ and E- with the OKT11 monoclonal antibody (anti-E-receptor antibody); (3) that E+ OKM1+ cells mediated both ADCC and NK activity; (4) that the phenotypes of PBMNC forming tumor cell conjugates were (a) OKM1+ (both E-receptor positive and negative) and (b) OKM1- E-receptor positive.     

B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms

B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser(338). We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser(445) was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser(445) phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser(445) primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition.     

Two distinct mechanisms of cytotoxicity by porcine alveolar macrophages in antibody-dependent and immobilized immune complex-dependent cellular cytotoxicity

The mechanisms of cytotoxicity by porcine pulmonary alveolar macrophages (PAM) involved in antibody-dependent cellular cytotoxicity (ADCC) and immobilized immune complex-dependent cellular cytotoxicity (IIC-DCC) were investigated. The results indicate that IIC-DCC was inhibited by both catalase and thioglycollate broth whereas these peroxide scavengers had no effect on ADCC in an 18-hr chromium-release assay. Furthermore, it was found that when the PAM and red blood cell targets were cross-linked with PHA, catalase still completely eliminated IIC-DCC and had no effect on ADCC, which suggests that catalase is able to penetrate the lytic site when the effector and targets are cross-linked as in ADCC. The presence of cytochalasin B, which inhibits internalization of immune complexes by PAM and presumably prevents intracellular killing, also had no effect on the differential susceptibility of IIC-DCC and ADCC to catalase. Finally, it is shown that the nonspecific cytotoxicity generated by exposing PAM to immune complexes in suspension in conjunction with cytochalasin B, so that the immune complex-bound Fc receptor (FcR) cannot be internalized, also was susceptible to catalase. These data show that the lytic mechanism involved in the nonspecific cytotoxicity generated by exposing PAM to immobilized immune complexes or immune complexes in suspension in conjunction with cytochalasin B, both of which prevent the internalization of immune complex-bound FcR, is mediated solely by peroxide whereas the lytic mechanism involved in ADCC operates, at least partially, through a peroxide-independent mechanism.     

Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron.

Phenanthroline, a strong iron chelator, prevents both the formation of DNA single-strand breaks and the killing of mouse cells produced by H2O2. These results, taken together with our previous findings, indicate that the DNA damage is produced by hydroxyl radicals formed when H2O2 reacts with chromatin-bound Fe2+ and that this damage is responsible for the killing effect.     

Polylysine activates and alters the divalent cation requirements of the insulin receptor protein tyrosine kinase

Protamine and poly(Lys) activate the protein tyrosine kinase of both the human placental insulin receptor and its purified recombinant cytoplasmic domain. Spermidine, poly(Arg) (average molecular mass 15 kDa), poly(Glu), Arg or Lys are not effective. Activation is stable, reversible, and optimal when the enzyme is preincubated with activator, divalent cation and ATP prior to the addition of exogenous protein substrates. The most striking feature of the activation is that it results in 20-30-fold stimulation of the kinase in the presence of 0.2-0.4 mM Mn2+ and induces equivalent activity in the presence of Mg2+ alone (0.4-4.0 mM). The activated protein tyrosine kinase has a specific activity (0.25-0.5 mumol/mg protein) that approaches that of well characterized protein serine kinases.