Carlos Rosales

Signal transduction during Fc receptor-mediated phagocytosis

Phagocytosis is the process whereby cells engulf large particles, usually over 0.5 μm in diameter. Phagocytosis is triggered by the interaction of opsonins that cover the particle to be internalized with specific receptors on the surface of the phagocyte. The best‐studied phagocytic receptors include the Fc receptors (FcR) that bind to the Fc portion of immunoglobulins. Cross‐linking of FcR on the phagocyte initiates a variety of signals, which lead through the reorganization of the actin cytoskeleton, and membrane remodeling, to the formation of the phagosome. From recent data, it is becoming clear that FcR‐mediated phagocytosis occurs as a series of steps that are regulated in a nonlinear manner and that signaling for phagocytosis does not terminate when the phagosome is formed. Several lipid molecules localize around the nascent phagosome and function as initiators of important signaling pathways for the late stages of phagolysosome formation. In addition, the use of particular signaling molecules may change for different receptors and may also vary depending on the activation or differentiation state of the cell. This review focuses on this new information and presents a model of our present understanding of the signal transduction events that regulate phagocytosis mediated by FcR.

Signal transduction by immunoglobulin Fc receptors.

Receptors for the Fc portion of immunoglobulin molecules (FcR) present on leukocyte cell membranes mediate a large number of cellular responses that are very important in host defense. Cross‐linking of FcR by immune complexes leads to functions such as phagocytosis, cell cytotoxicity, production and secretion of inflammatory mediators, and modulation of the immune response. Molecular characterization of FcRs indicates the existence of several types of these receptors, which seem to be redundant in their cell distribution and function. There is a great deal of interest in understanding how these various receptors signal the cell to respond in different ways during inflammation and the immune response. Previous studies indicate that FcR signaling shares elements with the T and B cell antigen receptors. Signaling is initiated in all of them by activation of tyrosine kinases of the Src and ZAP‐70 families. Subsequent events, which vary depending on the cell type and receptor involved, include activation of other enzymes such as phospholipase Cγ1, phosphatidylinositol‐3‐kinase, and mitogen‐activated protein kinase. Several recent lines of research, including studies of phagocytosis by FcR‐transfected cells, antibody‐dependent cytotoxicity by natural killer cells, mast cell degranulation, and FcR‐deficient mice, have given us new insights on the signal transduction pathways activated by FcRs. This review describes the advances in these areas and presents a general model for FcR‐mediated signaling. J. Leukoc. Biol. 63: 521–533; 1998.

Macrophage--Mycobacterium tuberculosis interactions: role of complement receptor 3.

Tuberculosis is the leading infectious disease in the world. Mycobacterium tuberculosis, the causal agent of this disease, invades macrophages and can replicate inside them. Because invasion of macrophages is a critical step for establishing a mycobacterial infection, there is much interest in understanding the mechanisms for M. tuberculosis entry into macrophages. Complement receptor 3 (CR3) is a heterodimeric surface receptor with multiple binding sites, which can mediate complement-opsonized as well as nonopsonic entrance of M. tuberculosis into macrophages. Here, we describe and discuss the role of CR3 in macrophage[bond]M. tuberculosis interactions. The actual information suggests that CR3 mediates a substantial amount of M. tuberculosis binding to macrophages, but CR3 is not related to the mechanisms that allow mycobacteria to survive and replicate intracellularly. Understanding the mechanisms of macrophage[bond]M. tuberculosis interaction will help developing more effective methods to prevent and treat tuberculosis in the future.

FCGAMMA RECEPTOR-MEDIATED MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION IN MONOCYTES IS INDEPENDENT OF RAS

Receptors for the Fc portion of immunoglobulin molecules (FcR) present on leukocyte cell membranes mediate a large number of cellular responses that are very important in host defense, including phagocytosis, cell cytotoxicity, production and secretion of inflammatory mediators, and modulation of the immune response. Cross-linking of FcR with immune complexes leads, first to activation of protein-tyrosine kinases. The molecular events that follow and that transduce signals from these receptors to the nucleus are still poorly defined. We have investigated the signal transduction pathway from Fc receptors that leads to gene activation and production of cytokines in monocytes. Cross-linking of FcR, on the THP-1 monocytic cell line, by immune complexes resulted in both activation of the transcription factor NF-κB and interleukin 1 production. These responses were completely blocked by tyrosine kinase inhibitors. In contrast, expression of dominant negative mutants of Ras and Raf-1, in these cells, did not have any effect on FcR-mediated nuclear factor activation, suggesting that the mitogen-activated protein kinase (MAPK) signaling pathway was not used by these receptors. However, MAPK activation was easily detected by in vitro kinase assays, after FcR cross-linking with immune complexes. Using the specific MAPK/extracellular signal-regulated kinase kinase (MAPK kinase) inhibitor PD98059, we found that MAPK activation is necessary for FcR-dependent activation of the nuclear factor NF-κB. These results strongly suggest that the signaling pathway from Fc receptors leading to expression of different genes important to leukocyte biology, initiates with tyrosine kinases and requires MAPK activation; but in contrast to other tyrosine kinase receptors, FcR-mediated MAPK activation does not involve Ras and Raf.

Phosphatidylinositol 3-kinase and extracellular signal-regulated kinase are recruited for Fc receptor-mediated phagocytosis during monocyte-to-macrophage differentiation

The molecular mechanism involved in Fc receptor‐mediated phagocytosis in the different cell types of the immune system is still poorly defined. We investigated the role of phosphatidylinositol 3‐kinase (PI 3‐K) and extracellular signal‐regulated kinase (ERK) in phagocytosis by monocytes and by monocyte‐differentiated macrophages. Peripheral blood monocytes and monocytic cells (THP‐1 cell line) were able to ingest IgG‐coated erythrocytes in the absence of additional stimulus. Phagocytosis by these cells was not blocked by wortmannin and LY294002, specific inhibitors of PI 3‐K, or by PD98059, a specific MEK/ERK inhibitor. However, upon differentiation of THP‐1 monocytes to macrophages, through treatment with retinoic acid and interferon‐γ (IFN‐γ), wortmannin and PD98059 blocked Fc receptor‐mediated phagocytosis efficiently. Inhibition of phagocytosis by PD98059 was observed after 24 h of IFN‐γ treatment, whereas wortmannin could inhibit phagocytosis only after 48 h of IFN‐γ treatment. Additionally, phagocytosis of IgG‐coated erythrocytes by neutrophils, a more efficient phagocyte, was inhibited by wortmannin and PD98059. Neutrophils and monocyte‐differentiated macrophages presented significantly more efficient phagocytosis than monocytes upon PMA stimulation. Taken together, these results indicate that poorly phagocytic leukocytes, such as monocytes, do not require PI 3‐K and ERK for phagocytosis. Upon differentiation into macrophages, however, ERK first and PI 3‐K second are recruited for regulation of phagocytosis. In addition, our data support the idea that professional phagocytes require ERK and PI 3‐K for efficient phagocytosis.

Phosphatidylinositol 3‐kinase and ERK are required for NF‐κB activation but not for phagocytosis

The molecular events that transduce signals from Fc receptors to the various cellular responses are still poorly defined. We have investigated the role of phosphatidylinositol 3‐kinase (PI 3‐K) and extracellular signal‐regulated kinase (ERK) in gene activation and phagocytosis in monocytes. In the THP‐1 monocytic cell line, cross‐linking of Fc receptors by immune complexes results in activation of the transcription factor NF‐κB, via activation of ERK. Activation of both ERK and NF‐κB was blocked by wortmannin and LY294002, specific inhibitors of PI 3‐K. Wortmannin also inhibited the Fc receptor‐mediated increase in the cytosolic calcium concentration, but it did not block immunoglobulin G (IgG)‐mediated phagocytosis. In addition, the ERK inhibitor PD98059 did not block phagocytosis of IgG‐coated erythrocytes. Both the increase in the cytosolic calcium concentration and phagocytosis depend on an active actin cytoskeleton, as indicated by the total lack of both responses after treatment with cytochalasin B. In contrast, cytochalasin B did not affect Fc receptor‐mediated activation of NF‐κB. These results identify PI 3‐K and ERK as important signaling molecules in the Fc receptor signal transduction pathway of monocytes, which leads to the nucleus for gene activation. These results also suggest that, in contrast to other cell types, unstimulated monocytes do not require PI 3‐K and ERK for phagocytosis.

Transmembrane Mutations to FcγRIIA Alter Its Association with Lipid Rafts: Implications for Receptor Signaling

Many immunoreceptors have been reported to associate with lipid rafts upon ligand binding. The way in which this association is regulated is still obscure. We investigated the roles for various domains of the human immunoreceptor FcγRIIA in regulating its association with lipid rafts by determining the resistance of unligated, or ligated and cross-linked, receptors to solubilization by the nonionic detergent Triton X-100, when expressed in RBL-2H3 cells. Deletion of the cytoplasmic domain, or destruction of the cytoplasmic palmitoylation site, had no effect on the association of the receptor with lipid rafts. A transmembrane mutant, A224S, lost the ability to associate with lipid rafts upon receptor cross-linking, whereas transmembrane mutants VA231-2MM and VVAL234-7GISF showed constitutive lipid raft association. Wild-type (WT) FcγRIIA and all transmembrane mutants activated Syk, regardless of their association with lipid rafts. WT FcγRIIA and mutants that associated with lipid rafts efficiently activated NF-κB, in an ERK-dependent manner. In contrast, WT FcγRIIA and the A224S mutant both presented efficient phagocytosis, while VA231-2MM and VVAL234-7GISF mutants presented lower phagocytosis, suggesting that phagocytosis may proceed independently of lipid raft association. These data identify the transmembrane domain of FcγRIIA as responsible for regulating its inducible association with lipid rafts and suggest that FcγRIIA-mediated responses, like NF-κB activation or phagocytosis, can be modulated by lipid raft association of the ligated receptor.

Neutrophils in Cancer: Two Sides of the Same Coin

Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs) have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions.

R‐Ras promotes tumor growth of cervical epithelial cells

R‐Ras is 55% identical to H‐Ras. However, these two oncogenes seem to have different tumor‐transforming potential. R‐Ras induced cell transformation in fibroblasts but not in other cell types. R‐Ras also reportedly induces a more invasive phenotype in breast epithelial cells through integrin activation. The authors studied the mechanisms whereby R‐Ras induces a malignant phenotype.

A recombinant vaccinia virus containing the papilloma E2 protein promotes tumor regression by stimulating macrophage antibody-dependent cytotoxicity

Abstract Human papillomavirus infection is associated with cervical cancer. The E6 and E7 papillomavirus proteins are normally required for the maintenance of the malignant phenotype. Expression of these proteins in infected cells is negatively regulated by the binding of the papilloma E2 protein to the long terminal control region of the papilloma virus genome. The E2 protein can also promote cell arrest and apoptosis in HeLa cells. Therefore, it is clear that this protein has the potential of inhibiting the malignant phenotype. Because, anticancer vaccines based in vaccinia viruses have recently been shown to be an effective way to treat and to eradicate tumors, a recombinant vaccinia virus expressing the E2 gene of bovine papilloma virus (Modified Vaccinia Ankara, MVA E2) was created, to explore further the antitumor potential of the E2 protein. A series of rabbits, containing the VX2 transplantable papilloma carcinoma, were treated with MVA E2. An impressive tumor regression, up to a complete disappearance of tumor, was observed in most animals (80%). In contrast, very little or no regression was detected if the normal vaccinia virus was used. Lymphocytes isolated from MVA E2-treated rabbits did not show cytotoxic activity against tumor cells. However, in these animals a humoral immune response against tumor cells was observed. These antitumor antibodies were capable of activating macrophages to destroy tumor cells efficiently. These data indicate that injecting the MVA E2 recombinant vaccinia virus directly into the tumor results in a robust and long-lasting tumor regression. Data also suggest that antitumor antibodies are responsible, at least in part, for eliminating tumors by activating macrophage antibody-dependent cytotoxicity.