Jeffrey V. Ravetch

Fc receptors: rubor redux.

Since their initial discovery over 30 years ago, cellular receptors for the Fc domain of immunoglobulins (FcRs) have posed something of an enigma for immunologists. These surface glycoproteins were known to be widely distributed on cells of the immune system, had specificity for different isotypes of immunoglobulin, and could mediate a variety of effector responses when cross-linked in vitro (Ravetch and Kinet, 1991). Molecular analysis of the genes and protein complexes that constitute these receptors had yielded a rich diversity of structures, revealing subunits, protein motifs, and signal transduction pathways shared with the more familiar immune receptors such as the antigen receptors on T cells and B cells (Keegan and Paul, 1992; Weiss and Littman, 1994). Yet, despite their ability to mediate a wide range of biological activities on isolated cells, ranging from antibody-dependent cellular cytoxicity to phagocytosis and mediator release from effector cells, the role of many of these receptors in vivo had remained obscure until relatively recently. The basic problem in determining their function in vivo was the redundancy of effector pathways triggered by immune complexes. Defined mutations were lacking in specific FcRs that would facilitate clarifying their contribution, as compared with other immunoglobulin-binding proteins like the complements, to the effector arm of an immune response. Several reports in the last 6 months, utilizing targeted gene disruption of many of the FcRs (Takai et al., 1994; Dombrowicz et al., 1993) have defined a critical and unexpected role for these receptors in the antibody-mediated inflammatory response. These studies demonstrated that F&s have a central role in initiating immunocomplex-triggered inflammation and are likely to be major contributors to the process of lymphocyte regulation of antibody production.

Cytotoxic antibodies trigger inflammation through Fc receptors

Pathogenic self-reactive antibodies are a significant cause of morbidity and mortality and contribute to both cytotoxic and immune complex-triggered inflammatory disorders, typified by rheumatic diseases, autoimmune hemolytic anemia, and thrombocytopenia. Roles have been proposed for Fc receptors, complement, and complement receptors in the pathogenesis of these disorders, although the contribution of each to autoimmune injury is unclear. gamma chain-deficient mice lacking Fc gamma RI and Fc gamma RIII are resistant to the development of experimental immune hemolytic anemia induced by polyclonal rabbit anti-mouse red blood cell IgG antibodies. This resistance is primarily a consequence of ineffective erythrophagocytosis, resulting from the lack of Fc gamma Rs on mononuclear phagocytes. Similarly, gamma chain-deficient mice are completely resistant to the development of experimental immune thrombocytopenia induced by mouse anti-platelet antibodies. These data suggest that Fc receptors play an integral role in the pathogenesis of type II hypersensitivity and suggest potential therapeutic benefits of Fc receptor blockade.

A Dominant Role for Mast Cell Fc Receptors in the Arthus Reaction

Antibody-antigen complexes are central to the inflammatory response and are implicated in the development of such diverse diseases as systemic lupus erythematosis, rheumatoid arthritis, immune glomerulonephritis, and vasculitis. We recently demonstrated that experimental immune complex-mediated injury in mice, as modeled by the cutaneous Arthus reaction, requires receptors for the Fc portion of the antibody and is unaffected by deficiencies in complement components. However, the responsible cell type(s) and Fc receptor(s) were not known. We now demonstrate by differential reconstitution in vivo that Fc gamma RIII on mast cells is necessary for this inflammatory response. We propose a general model of antibody-mediated diseases as an immunopathologic spectrum whose specific manifestations are determined by the Fc receptor and cell type engaged.

Molecular Determinants of the Myristoyl-electrostatic Switch of MARCKS

MARCKS is a protein kinase C (PKC) substrate which binds calcium/calmodulin and actin, and which has been implicated in cell motility, phagocytosis, membrane traffic, and mitogenesis. MARCKS cycles on and off the membrane via a myristoyl electrostatic switch (McLaughlin, S., and Aderem, A. (1995) Trends Biochem. Sci. 20, 272-276). Here we define the molecular determinants of the myristoyl-electrostatic switch. Mutation of the N-terminal glycine results in a nonmyristoylated form of MARCKS which does not bind membranes and is poorly phosphorylated. This indicates that myristic acid targets MARCKS to the membrane, where it is efficiently phosphorylated by PKC. A chimeric protein in which the N terminus of MARCKS is replaced by a sequence, which is doubly palmitoylated, is phosphorylated by PKC but not released from the membrane. Thus two palmitic acid moieties confer sufficient membrane binding energy to render the second, electrostatic membrane binding site superfluous. Mutation of the PKC phosphorylation sites results in a mutant which does not translocate from the membrane to the cytosol. A mutant in which the intervening sequence between the myristoyl moiety and the basic effector domain is deleted, is not displaced from the membrane by PKC dependent phosphorylation, fulfilling a theoretical prediction of the model. In addition to the nonspecific membrane binding interactions conferred by the myristoyl-electrostatic switch, indirect immunofluorescence microscopy demonstrates that specific protein-protein interactions also specify the intracellular localization of MARCKS.

Transcription mapping of a 100 kb locus of Plasmodium falciparum identifies an intergenic region in which transcription terminates and reinitiates.

We have mapped Plasmodium falciparum erythrocytic stage transcription units on chromosome 10 in the vicinity of the gene encoding the glycophorin binding protein (GBP130) using yeast artificial chromosomes (YACs). Three erythrocytic stage transcription units are clustered in a 40 kb region. Two of these genes are closely linked, separated by less than 2 kb. Nuclear run‐on data demonstrate that transcription of these two genes, though unidirectional, is monocistronic. Within this intergenic region are the sites at which transcription of the upstream gene terminates and the GBP130 gene initiates. These studies represent the first description of the minimal and necessary cis‐acting elements for transcription termination and initiation in this protozoan parasite.

PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc gamma RIIIA promoter.

Expression of the low‐affinity Fc receptor for IgG (murine Fc gamma RIIIA) is restricted to cells of myelomonocytic origin. We report here the promoter structure, the proximal DNA sequences responsible for transcription of Fc gamma RIIIA in macrophages and the protein factors which interact with these sequences. A 51 bp sequence, termed the myeloid restricted region (MRR), was both necessary and sufficient for conferring cell type‐specific expression in macrophages. Reporter constructs containing mutations in this sequence result in the loss of MRR activity upon transfection into the macrophage cell line, RAW264.7. Two cis‐acting elements have been identified and are required for full promoter function. These same elements analyzed by EMSA define two binding sites recognized by nuclear factors derived from macrophages. A 3′ purine tract (‐50 to ‐39) within the MRR binds the macrophage and B cell‐specific factor, PU.1, and a second E box‐like element, termed MyE, upstream of the PU.1 box (‐88 to ‐78) binds the HLH factors TFE3 and USF. EMSA studies using RAW cell extracts suggest that both PU.1 and MyE factors may bind simultaneously to the MRR resulting in a ternary complex that is responsible, in part, for the myeloid‐specific activity of the Fc gamma RIIIA promoter.

A tandemly repeated sequence determines the binding domain for an erythrocyte receptor binding protein of P. falciparum

Erythrocyte invasion by the malarial merozoite is a receptor-mediated process, an obligatory step in the development of the parasite. The Plasmodium falciparum protein GBP-130, which binds to the erythrocyte receptor glycophorin, is shown here to encode the binding site in a domain composed of a tandemly repeated 50 amino acid sequence. The amino acid sequence of GBP-130, deduced from the cloned and sequenced gene, reveals that the protein contains 11 highly conserved 50 amino acid repeats and a charged N-terminal region of 225 amino acids. Binding studies on recombinant proteins expressing different numbers of repeats suggest that a correlation exists between glycophorin binding and repeat number. Thus, a repeat domain, a common feature of plasmodial antigens, has been shown to have a function independent of the immune system. This conclusion is further supported by the ability of antibodies directed against the repeat sequence to inhibit the in vitro invasion of erythrocytes by merozoites.

A YAC contig map of plasmodium falciparum chromosome 4: Characterization of a DNA amplification between two recently separated isolates

We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen.