Randall G. Worth

Differential kinase requirements in human and mouse Fc‐gamma receptor phagocytosis and endocytosis

Fc gamma receptors (FcγRs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (FcγR) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src‐related tyrosine kinases (SRTKs) and phosphatidyl inositol 3‐kinases (PI3K) play important roles in FcγR phagocytosis. Herein, we demonstrate that these kinases are not required for FcγR endocytosis. Endocytosis of heat‐aggregated IgG (HA‐IgG) by COS‐1 cells stably transfected with FcγRIIA or chimeric FcγRI‐γ‐γ (EC‐TM‐CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG‐sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow‐derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when FcγRIIA was cross‐linked using HA‐IgG or EA. However, inhibition of FcγR phosphorylation prevented only FcγR phagocytosis. Finally, biochemical analyses of PI3K(p85)‐Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during FcγR phagocytosis and endocytosis. Overall, our results indicate that FcγR endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition‐based model in which PI3K and c‐Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.

Interactions of integrins with their partner proteins in leukocyte membranes

Integrins participate in many aspects of immunologic and inflammatory responses, especially those involving cell migration, adherence, and activation. Although leukocyte integrins such as complement receptor type 3 (CR3) are known to carry out certain functions without the intervention of other plasma membrane receptors, many plasma membrane proteins are now known to physically interact and functionally cooperate with integrins. Several of these interactions are highly dynamic within cell membranes; thus integrin-partner protein interactions change during certain physiological processes. This allows an extraordinary adaptability of the system to prime and promote proinflammatory signaling. Since our discovery of the CR 3-FcyRIIIB interaction, the plasma membrane protein repertoire of β1, β2, and β3 integrins has grown to include: FcγRIIA (CD32), uPAR (urokinase-type plasminogen activator receptor; CD87), CD14, voltage-gated K+ channels (Kvl.3), integrin-associated protein (IAP), CD98, tetraspans (TM4SF), insulin receptors, and PDGFβ receptors. In this article we will highlight certain features of this growing field of research, especially with regard to their relevance in immunology and inflammation.

Signal sequence within FcγRIIA controls calcium wave propagation patterns: Apparent role in phagolysosome fusion

Calcium oscillations and traveling calcium waves have been observed in living cells, although amino acid sequences regulating wave directionality and downstream cell functions have not been reported. In this study we identify an amino acid sequence within the cytoplasmic domain of the leukocyte IgG receptor FcγRIIA that affects the amplitude of calcium spikes and the spatiotemporal dynamics of calcium waves in the vicinity of phagosomes. By using high-speed microscopy to map calcium-signaling routes within cells, we have discovered that bound IgG-coated targets trigger two calcium waves traveling in opposite directions about the perimeter of cells expressing FcγRIIA. After phagocytosis, one calcium wave propagates around the plasma membrane to the site of phagocytosis where it splits into two calcium signals: one traveling to and encircling the phagosome once, and the second continuing around the plasma membrane to the point of origin. However, in a genetically engineered form of FcγRIIA containing a mutation in the cytoplasmic L-T-L motif, the calcium signal travels around the plasma membrane, but is not properly routed to the phagosome. Furthermore, these calcium pattern-deficient mutants were unable to support phagolysosome fusion, although recruitment of phagolysosome-associated proteins lysosome-associated protein 1, Rab5, and Rab7 were normal. Our findings suggest that: (i) calcium signaling is a late step in phagolysosome fusion, (ii) a line of communication exists between the plasma membrane and phagosome, and (iii) the L-T-L motif is a signal sequence for calcium signal routing to the phagosome.

Identification of a Novel Mode of Complement Activation on Stimulated Platelets Mediated by Properdin and C3(H2O)

Elevated numbers of activated platelets circulate in patients with chronic inflammatory diseases, including atherosclerosis and coronary disease. Activated platelets can activate the complement system. Although complement activation is essential for immune responses and removal of spent cells from circulation, it also contributes to inflammation and thrombosis, especially in patients with defective complement regulation. Proinflammatory activated leukocytes, which interact directly with platelets in response to vascular injury, are among the main sources of properdin, a positive regulator of the alternative pathway. The role of properdin in complement activation on stimulated platelets is unknown. Our data show that physiological forms of human properdin bind directly to human platelets after activation by strong agonists in the absence of C3, and bind nonproportionally to surface CD62P expression. Activation of the alternative pathway on activated platelets occurs when properdin is on the surface and recruits C3b or C3(H2O) to form C3b,Bb or a novel cell-bound C3 convertase [C3(H2O),Bb], which normally is present only in the fluid phase. Alternatively, properdin can be recruited by C3(H2O) on the platelet surface, promoting complement activation. Inhibition of factor H–mediated cell surface complement regulation significantly increases complement deposition on activated platelets with surface properdin. Finally, properdin released by activated neutrophils binds to activated platelets. Altogether, these data suggest novel molecular mechanisms for alternative pathway activation on stimulated platelets that may contribute to localization of inflammation at sites of vascular injury and thrombosis.

IgG-complex stimulated platelets: a source of sCD40L and RANTES in initiation of inflammatory cascade.

Platelets are a crucial element in maintenance of hemostasis. Other functions attributable to platelets are now being appreciated such as their role in inflammatory reactions and vascular remodeling. Platelets have been reported to bind immunological stimuli like IgG-complexes and the understanding that platelets may participate in immunological reactions has been speculated for nearly 50years. In previous observations, we demonstrated that platelets could bind and internalize aggregated IgG-complexes without inducing platelet aggregation or granule release. To characterize this observation further, we tested the hypothesis that aggregated IgG-complexes do not activate platelets. To this end, platelets were stimulated with IgG-complexes or thrombin as a positive control and evaluated for activation by aggregation, expression of surface markers and production of cytokines. Activation with thrombin resulted in aggregation, expression of high levels of CD62P (P-selectin) expression and activation of the fibrinogen receptor, alpha(IIb)beta(3). Furthermore, stimulation with thrombin resulted in significant amounts of sCD40L (CD154) and RANTES (CCL5). However, platelets stimulated with IgG-complexes resulted in no aggregation and low levels of CD62P expression. Surprisingly, platelets stimulated with aggregated IgG-complexes released similar amounts of sCD40L and RANTES as platelets activated by thrombin. These data suggest that platelets are capable of secreting inflammatory molecules in response to IgG-complexes.

The monocyte Fcγ receptors FcγRI/γ and FcγRIIA differ in their interaction with Syk and with Src‐related tyrosine kinases

There are important differences in signaling between the Fc receptor for immunoglobulin G (IgG) FcγRIIA, which uses the Ig tyrosine‐activating motif (ITAM) within its own cytoplasmic domain, and FcγRI, which transmits signals by means of an ITAM located within the cytoplasmic domain of its associated γ‐chain. For example, in transfected epithelial cells and COS‐1 cells, FcγRIIA mediates phagocytosis of IgG‐coated red blood cells more efficiently than does FcγRI/γ, and enhancement of phagocytosis by Syk kinase is more pronounced for FcγRI/γ than for FcγRIIA. In addition, structure/function studies indicate that the γ‐chain ITAM and the FcγRIIA ITAM have different requirements for mediating the phagocytic signal. To study the differences between FcγRIIA and FcγRI/γ, we examined the interaction of FcγRIIA and the FcγRI/γ chimera FcγRI‐γ‐γ (extracellular domain–transmembrane domain–cytoplasmic domain) with Syk kinase and with the Src‐related tyrosine kinases (SRTKs) Hck and Lyn in transfected COS‐1 cells. Our data indicate that FcγRIIA interacts more readily with Syk than does FcγRI‐γ‐γ and suggest that one consequence may be the greater phagocytic efficiency of FcγRIIA compared with FcγRI/γ. Furthermore, individual SRTKs affect the efficiency of phagocytosis differently for FcγRI‐γ‐γ and FcγRIIA and also influence the ability of these receptors to interact with Syk kinase. Taken together, the data suggest that differences in signaling by FcγRIIA and FcγRI‐γ‐γ are related in part to interaction with Syk and Src kinases and that individual SRTKs play different roles in FcγR‐mediated phagocytosis.

Internalization of IgG-Coated Targets Results in Activation and Secretion of Soluble CD40 Ligand and RANTES by Human Platelets

ABSTRACT Platelets are crucial elements for maintenance of hemostasis. Other functions attributable to platelets are now being appreciated, such as their role in inflammatory reactions and host defense. Platelets have been reported to bind immunological stimuli like IgG complexes, and for nearly 50 years it has been speculated that platelets may participate in immunological reactions. Platelets have been reported to bind and internalize various substances, similar to other leukocytes, such as macrophages and dendritic cells. In the present study, we tested the hypothesis that human platelets can bind and internalize IgG-coated particles, similar to leukocytes. To this end, we observed that interaction with IgG-coated beads resulted in platelet activation (as measured by CD62P expression), internalization of targets, and significant soluble CD40 ligand (sCD40L) and RANTES (regulated upon activation, normal T cell expresses and secreted) secretion. Blocking FcγRIIA with monoclonal antibody (MAb) IV.3 or inhibiting actin remodeling with cytochalasin D inhibited platelet activation, internalization, and cytokine production. These data suggest that platelets are capable of mediating internalization of IgG-coated particles, resulting in platelet activation and release of both sCD40L and RANTES.

Cancer and Thrombosis: The Platelet Perspective

Platelets are critical to hemostatic and immunological function, and are key players in cancer progression, metastasis, and cancer-related thrombosis. Platelets interact with immune cells to stimulate anti-tumor responses and can be activated by immune cells and tumor cells. Platelet activation can lead to complex interactions between platelets and tumor cells. Platelets facilitate cancer progression and metastasis by: (1) forming aggregates with tumor cells; (2) inducing tumor growth, epithelial-mesenchymal transition, and invasion; (3) shielding circulating tumor cells from immune surveillance and killing; (4) facilitating tethering and arrest of circulating tumor cells; and (5) promoting angiogenesis and tumor cell establishment at distant sites. Tumor cell-activated platelets also predispose cancer patients to thrombotic events. Tumor cells and tumor-derived microparticles lead to thrombosis by secreting procoagulant factors, resulting in platelet activation and clotting. Platelets play a critical role in cancer progression and thrombosis, and markers of platelet-tumor cell interaction are candidates as biomarkers for cancer progression and thrombosis risk.

Capsule Influences the Deposition of Critical Complement C3 Levels Required for the Killing of Burkholderia pseudomallei via NADPH-Oxidase Induction by Human Neutrophils

Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD50 via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.

A novel conditional platelet depletion mouse model reveals the importance of platelets in protection against Staphylococcus aureus bacteremia

Platelets are critical cells for maintaining vascular hemostasis, but their activities in other processes are becoming apparent. Specifically, the ability of platelets to recognize and respond to infectious agents is an important area of investigation. To understand the physiologic roles of platelets in vivo, most researchers have used antibody‐mediated platelet depletion, which has certain limitations.