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Research
Molecular Inflammation Section
Juan Rivera, Ph.D.
Section Chief, Molecular Inflammation Section
Molecular Immunology and Inflammation Branch (MIIB)
Phone: (301) 496-7592
Fax: (301) 402-0012
E-mail: riveraj@mail.nih.gov
Research Overview
Autoimmune diseases, asthma, and chronic allergies result from dysfunctions in the regulation of the immune system. The work in this laboratory is directed towards understanding how allergenic agents and immune complexes activate one particular family of receptors (Fc receptors) and, through them, ultimately control the genes that produce the signals (cytokines) that in turn recruit other cells in the inflammatory response. Understanding this system is essential for developing targeted therapeutic agents that can ameliorate immune system disorders with minimal side effects.
A fundamental problem in receptor biology is understanding how the strength or duration of a stimulus causes a receptor to engage downstream effectors in a manner that can result in different cellular responses. The implication is that by varying the strength or duration of a stimulus a receptor can differentially engage downstream effectors and thereby include or exclude signaling molecules needed for a particular cellular response.
Recently the importance of the architectural framework for signal transduction has become better understood as adaptor and scaffold signal molecules are discovered. These molecules serve to assemble signaling molecules into functional units. They may also determine what downstream effector pathways are engaged by inclusion or exclusion of certain molecules in these complexes, which may result from the strength and type of stimulus. Specificity is conferred not just by particular enzymes in signaling pathways, but by structural constraints within the whole signaling complex arising from portions of enzymes or from other macromolecules with no apparent enzymatic activity. Understanding of signaling architecture is still in its infancy.
The primary objective of this laboratory is to explore receptor-activated molecular mechanisms that regulate gene expression, using the mast cell as a model. The mast cell is a white blood cell that stores certain chemicals (e.g. histamines) in granules for immediate release when stimulated, and generates other chemicals (including cytokines) when genes are subsequently turned on. These chemicals in turn act as signals to other cells. The mast cell is a key player in the systemic response of organisms to inflammatory agents -- or allergens -- such as pollen, dust mites, and insect venom that provoke an inflammatory response in sensitive individuals. However, these cells also play an important role in defending an individual against certain pathogenic parasitic and bacterial insults. In this case the localized inflammatory response is beneficial as it serves to clear the offending agent.
The systemic response begins with circulating allergens that form a complex with IgE or IgG antibodies already bound to the high-affinity Fc receptor for IgE-FcεRI or the low affinity Fc receptor for IgG-FcγRIII on the mast cells. This complex formation sets in motion a cascade of molecular signals that ultimately modulate gene expression, resulting in the production and secretion of a diverse array of cytokines. Cytokines are soluble glycoproteins (molecules with sugars and proteins) that are released by immune system cells and act to regulate immune responses by binding specific receptors but without actively catalyzing reactions. The different cytokines both reflect the cellular and molecular environments producing them and send signals that recruit different responses in other immune cells.
Some of the studies of this Section are focused on detailing the initial events after the stimulation of FcεRI. One model of these initial events developed from studies in the Chemical Immunology Section (see this Web site for details) has gained support from the studies by Dr. M. Kovarova, a new member of the Molecular Inflammation Section. The evidence supports a model in which FcεRI phosphorylation (activation) can occur independent of specialized plasma membrane domains called lipid rafts. This contrasts with a second model that suggests lipid rafts co-localize the receptor with the activating kinase, Lyn. Nevertheless, recent results in this laboratory support a role for lipid rafts in events immediately downstream from the phosphorylation (activation) of FcεRI.
Furthermore, to clarify events immediately downstream from the Fc receptors, the Molecular Inflammation Section studies the contribution of architectural frameworks and the control of cytokine gene expression by researching the function of some recently identified signaling proteins, all expressed exclusively in hematopoietic cells: i.e. the linker for activation of T cells (LAT) and the guanine nucleotide exchange factor Vav1. The role of these signaling proteins is evaluated using mast cell cytokine production and, where possible, the in vivo activation of mast cells in knockout mice that do not express these proteins.
LAT was found to be an essential component of IgE-FcεRI receptor signaling and, thus, of allergy conduction. The LAT protein serves as a scaffold that assembles other signaling proteins and also links the FceRI receptor to regulation of mast cell degranulation. LAT knockout mice do not increase their serum histamine levels when exposed to allergens as found in wild type (normal) mice. This lack of histamine was shown to be due to an inability of mast cells to release their granule content, rather than a reduction in the number of mast cells. Moreover, the failure to degranulate is linked to an inability to mobilize intracellular calcium stores.
Phosphorylation of three important regulators of intracellular calcium stores are reduced in LAT-deficient mice; these are the adaptor protein SLP-76 and two isoforms of the inositol phosphate-generating phospholipase C (PLCg 1 and PLCg 2). Calcium is necessary not just for degranulation, but for the activation of many transcription factors. Additional analysis showed that this defect in calcium mobilization through MAP kinases also prevents the expression of cytokine genes. In vitro experiments showed that in the LAT-deficient mast cells there was deficient production of the mRNAs for many cytokines, including three interleukin isoforms (IL-2, IL-3, and IL-10); other cytokines (IL-4, IL-6, and TNF-alpha) were less severely affected.
Vav1 is a guanine nucleotide exchange factor, a protein that catalyzes the release of guanine diphosphate (GDP) from certain small GTP-binding proteins of the Rho family GTPases so that the high-energy form, guanine triphosphate (GTP) can be bound. This initiates GTP hydrolysis, an energy-generating step important to facilitating the rearrangement of the cell actin cytoskeleton and the cell response. In vivo studies on knockout mice demonstrated that Vav1-deficiency diminished the in vivo mast cell degranulation, but the response remained stronger than in mast cells from LAT-deficient mice. In vitro studies show that Vav1 controls the activation of two isoforms of phospholipase C gamma, a protein which functions to generate a product (inositol 1, 4, 5-trisphosphate) that increases the intracellular calcium concentration.
PLC-γ contains SH2 domains (Src homology domain 2) that allow it to interact with tyrosine phosphorylated receptor-tyrosine kinases (RTKs). This allows PLC-gamma to be intimately associated with the signal transduction complexes of the membrane as well as membrane phospholipids that are its substrates. Activation of PLC-γ leads primarily to the hydrolysis of membrane phosphatidylinositol bisphosphate (PIP2) leading to an increase in intracellular diacylglycerol (DAG) and inositol trisphosphate (IP3). The released IP3 interacts with intracellular membrane receptors leading to an increased release of stored calcium ions. Together, the increased DAG and intracellular free calcium ion concentrations lead to increased activity of PKC.
Other laboratories previously showed that Vav1, once phosophorylated, activates Rac and Ras members of the Rho family of GTPases. This family of molecular switches controls a wide range of signaling pathways through the cytoskeletal scaffolding system, lipid signals through the lipid kinase, phosphoinositide 3-kinase (PI 3-kinase), and the activation of MAP kinase pathways. Rac also stimulates degranulation through the IP3-calcium pathway. It is apparently through this route that Vav1 regulates the intracellular calcium responses required for effective mast cell activation upon stimulation of the FcεRI receptor.
Immunoprecipitation studies reveal that LAT, Vav1, SLP-76, and Grb2, (another SH2 domain-containing protein) precipitate together, indicating that they are components in a common macromolecular signaling complex. Because LAT is a protein which has been lipid modified (palmitolylated), it could serve as a backbone for a complex signal transduction assembly in lipid rafts. Biochemical isolation of lipid rafts, along with confocal laser microscopy, showed that stimulation of FceRI receptors acted to recruit these receptors and Vav1 into lipid rafts that also contained LAT, Grb2, and the small Rho GTPase, Rac1. In the absence of Vav1, the activity of one MAP kinase, JNK1 was impaired, but activity of a different MAP kinase, ERK2, was enhanced, suggesting that Vav1 helps regulate a specific subset of the activities of the LAT-organized signaling complex.
In summary, proteins like LAT and Vav1, were found to be important components of the IgE FcεRI receptor response to stimulation. LAT is a key architectural component for bringing other proteins together. In its absence there are major deficiencies in mast cell degranulation, the immediate response to receptor stimulation, and the longer-term cytokine response that signals other cells. Though the absence of Vav1 has a more limited effect than the lack of LAT, it also regulates the expression of specific cytokines. Because these proteins occur only in hematopoetic cells, they are important as potential therapeutic targets. This laboratory will continue to explore potential targets for drug intervention by defining the regions of these proteins that determine their specific activity and by identifying other proteins that interact with them.
Selected Publications
Gonzalez-Espinosa C, Odom S, Olivera A, Hobson JP, Martinez MEC, Oliveira-dos-Santos A, Barra L, Spiegel S, Penninger J, Rivera J. Weak stimulation of the high affinity IgE receptor on mast cells causes preferential signaling and induction of selected lymphokines. FASEB J. 2003; 17 (7): C11-C11 Suppl. S.
Parravicini V, Gadina M, Kovarova M, Odom S, Gonzalez-Espinosa C, Furumoto Y, Saitoh S, Samelson LE, O'Shea JJ, Rivera J. Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat Immunol. 2002; 3(8):741-8.
Rivera J, Arudchandran R, Gonzalez-Espinosa C, Manetz TS, Xirasagar S. A perspective: regulation of Ige receptor-mediated mast cell responses by a Lat-organized plasma membrane-localized signaling complex. Int Arch Allergy Immunol. 2001; 124(1-3): 137-41.
Manetz TS, Gonzalez-Espinoza C, Arudchandran R, Xirasagar S, Tybulewicz V, Rivera J. Vav1 is an essential regulator of PLC\gamma and calcium responses in mast cells. Mol Cell Biol. 2001; 21: 3763-74.
Liu Y, Graham C, Parravicini V, Brown M J, Rivera J, Shaw S. Protein kinase C-\theta (PKC \theta) is expressed in mast cells and is functionally involved in FcRI signaling. J Leukocyte Biol. 2001; 69: 831-40.
See complete list of publications
Updated September 17, 2007
