Physical Constraints and Forces Involved in Phagocytosis.

Phagocytosis is a specialized process that enables cellular ingestion and clearance of microbes, dead cells and tissue debris that are too large for other endocytic routes. As such, it is an essential component of tissue homeostasis and the innate immune response, and also provides a link to the adaptive immune response. However, ingestion of large particulate materials represents a monumental task for phagocytic cells. It requires profound reorganization of the cell morphology around the target in a controlled manner, which is limited by biophysical constraints. Experimental and theoretical studies have identified critical aspects associated with the interconnected biophysical properties of the receptors, the membrane, and the actin cytoskeleton that can determine the success of large particle internalization. In this review, we will discuss the major physical constraints involved in the formation of a phagosome. Focusing on two of the most-studied types of phagocytic receptors, the Fcγ receptors and the complement receptor 3 (αMß2 integrin), we will describe the complex molecular mechanisms employed by phagocytes to overcome these physical constraints.

HGAL localization to cell membrane regulates B-cell receptor signaling.

Human germinal center-associated lymphoma (HGAL) is specifically expressed only in germinal center (GC) B lymphocytes and GC-derived lymphomas. HGAL protein decreases lymphocyte motility by inhibiting the ability of myosin to translocate actin via direct interaction with F-actin and myosin II and by activating RhoA signaling via direct interactions with RhoA-specific guanine nucleotide exchange factors. HGAL protein also regulates B-cell receptor (BCR) signaling by directly binding to and enhancing Syk kinase activity and activation of its downstream effectors. Herein we demonstrate that HGAL protein can be myristoylated and palmitoylated and that these modifications localize HGAL to cellular membrane raft microdomains with distinct consequences for BCR signaling and chemoattractant-induced cell mobility. In BCR signaling, raft localization of HGAL facilitates interaction with Syk and modulation of the BCR activation and signaling, which induces HGAL phosphorylation and redistribution from lipid raft to bulk membrane and cytoplasm, followed by degradation. In contrast, HGAL myristoylation and palmitoylation avert its inhibitory effects on chemoattractant-induced cell motility. These findings further elucidate the growing and complex role of HGAL in B-cell biology and suggest that membrane-bound and cytoplasmic HGAL protein differently regulates distinct biological processes.

Autotaxin produced by stromal cells promotes LFA-1-independent and Rho-dependent interstitial T cell motility in the lymph node paracortex.

T cells exhibit high-speed migration within the paracortical T zone of lymph nodes (LNs) as they scan cognate Ags displayed by dendritic cells in the tissue microenvironment supported by the network of stromal cells. Although intranodal T cell migration is controlled in part by chemokines and LFA-1/ICAM-1, the mechanisms underlying their migratory activity independent of these factors remain to be elucidated. In this study, we show that LN stromal cells constitutively express autotaxin (ATX), an ectoenzyme that is important for the generation of lysophosphatidic acid (LPA). Importantly, CCL21(+) stromal cells in the T zone produced and immobilized ATX on their cell surface. Two-photon imaging using LN tissue slices revealed that pharmacological inhibition of ATX or LPA receptors significantly reduced T cell migration, and this was further exacerbated by blockage of Gαi signaling or LFA-1. Therefore, T cell motility mediated by the ATX-LPA axis was independent of Gαi and LFA-1. LPA induced slow intermittent movement of T cells in vitro in a LFA-1-independent manner and enhanced CCL21-induced migration. Moreover, LPA and CCL21 cooperatively augmented RhoA activity in T cells, which was necessary for efficient intranodal T cell migration via the downstream ROCK-myosin II pathway. Taken together, T zone stromal cells control optimal migratory behavior of T cells via multiple signaling cues mediated by chemokines and ATX/LPA.

Controversy and consensus regarding myosin II function at the immunological synapse.

Regulated actin dynamics play a central role in modulating signaling events at the immunological synapse (IS). Polymerization of actin filaments at the periphery of the IS, coupled to depolymerization near the center, generates a centripetal flow of the actin network and associated movement of signaling molecules. A recent flurry of papers addresses the role of myosin II in facilitating these events. Investigators agree that myosin II is present at the IS, where it forms actomyosin arcs within the peripheral supramolecular activation cluster, a region corresponding to the lamellum of migrating cells. However, there is substantial disagreement about the extent to which myosin II drives IS formation and signaling events leading to T cell activation.

The human cytokine TSLP triggers a cell-autonomous dendritic cell migration in confined environments.

Dendritic cells (DCs) need to migrate in the interstitial environment of peripheral tissues to reach secondary lymphoid organs and initiate a suitable immune response. Whether and how inflamed tissues instruct DCs to emigrate is not fully understood. In this study, we report the unexpected finding that the epithelial-derived cytokine TSLP triggers chemokinesis of resting primary human DCs in a cell-autonomous manner. TSLP induced the polarization of both microtubule and actin cytoskeletons and promoted DC 3-dimensional migration in transwell as well as in microfabricated channels that mimic the confined environment of peripheral tissues. TSLP-induced migration relied on the actin-based motor myosin II and was inhibited by blebbistatin. Accordingly, TSLP triggered the redistribution of phosphorylated myosin II regulatory light chain to the actin cortex, indicating that TSLP induces DC migration by promoting actomyosin contractility. Thus, TSLP produced by epithelial cells in inflamed tissue has a critical function in licensing DCs for cell-autonomous migration. This indicates that cytokines can directly trigger cell migration, which has important implications in immune physiopathology and vaccine design.

CXCL13/CXCR5 signaling enhances BCR-triggered B-cell activation by shaping cell dynamics.

Continuous migration of B cells at the follicle contrasts with their stable arrest after encounter with antigen. Two main ligand/receptor pairs are involved in these cell behaviors: the chemokine CXCL13/chemokine receptor CXCR5 and antigen/BCR. Little is known regarding the interplay between CXCR5 and BCR signaling in the modulation of B-cell dynamics and its effect on B-cell activation. We used a 2-dimensional model to study B-cell migration and antigen recognition in real time, and found that BCR signaling strength alters CXCL13-mediated migration, leading to a heterogeneous B-cell behavior pattern. In addition, we demonstrate that CXCL13/CXCR5 signaling does not impair BCR-triggered immune synapse formation and that CXCR5 is excluded from the central antigen cluster. CXCL13/CXCR5 signaling enhances BCR-mediated B-cell activation in at least 2 ways: (1) it assists antigen gathering at the synapse by promoting membrane ruffling and lymphocyte function-associated antigen 1 (LFA-1)-supported adhesion, and (2) it allows BCR signaling integration in motile B cells through establishment of LFA-1-supported migratory junctions. Both processes require functional actin cytoskeleton and non-muscle myosin II motor protein. Therefore, the CXCL13/CXCR5 signaling effect on shaping B-cell dynamics is an effective mechanism that enhances antigen encounter and BCR-triggered B-cell activation.

Target-specific mechanics of phagocytosis: protrusive neutrophil response to zymosan differs from the uptake of antibody-tagged pathogens.

The physical mechanisms that control target-specific responses of human neutrophils to distinct immune threats are poorly understood. Using dual-micropipette manipulation, we have quantified and compared the time courses of neutrophil phagocytosis of two different targets: zymosan (a prominent model of fungal infection), and antibody-coated (Fc) particles. Our single-live-cell/single-target approach exposes surprising differences between these two forms of phagocytosis. Unlike the efficient uptake of 3-µm Fc targets (within ~66 seconds), the engulfment of similarly sized zymosan is slow (~167 seconds), mainly due to the formation of a characteristic pedestal that initially pushes the particle outwards by ~1 µm. Despite a roughly twofold difference in maximum cortical tensions, the top 'pull-in' speeds of zymosan and Fc targets are indistinguishable at ~33 nm/second. Drug inhibition shows that both actin as well as myosin II partake in the regulation of neutrophil cortical tension and cytoplasmic viscosity; other than that, myosin II appears to play a minor role in both forms of phagocytosis. Remarkably, an intact actin cytoskeleton is required to suppress, in antibody-mediated phagocytosis, the initially protrusive deformation that distinguishes the neutrophil response to zymosan.

Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.

Early complement factors in the local tissue immunocomplex generated during intestinal ischemia/reperfusion injury.

Recent work reveals that the innate immune system is able to recognize self-targets and initiate an inflammatory response similar to that of pathogens. One novel example of this innate autoimmunity is ischemia/reperfusion (I/R) injury, in which reperfusion of the ischemic tissues elicits an acute inflammatory response activated by natural IgM (nIgM) binding to ischemia-specific self-antigens, which are non-muscle myosin heavy chains type II (NMHC-II) subtype A and C. Subsequently, the complement lectin pathway is activated and eventually tissue injury occurs. Although earlier studies in the intestinal model showed that the classical complement pathway did not initiate I/R injury, C1q deposition was still observed in the local injured tissues by imaging analysis. Moreover, the involvement of the alternative complement pathway became unclear due to conflicting reports using different knockout mice. To explore the immediate downstream pathway following nIgM-ischemic antigen interaction, we isolated the nIgM-ischemic antigen immunocomplexes from the local tissue of animals treated in the intestinal I/R injury model, and examined the presence of initial molecules of three complement pathways. Our results showed that mannan-binding lectin (MBL), the early molecule of the lectin pathway, was present in the nIgM-ischemic Ag immunocomplex. In addition, C1q, the initial molecule of the classical pathway was also detected on the immunocomplex. However, Factor B, the early molecule in the alternative pathway, was not detected in the immunocomplex. To further examine the role of the alternative pathway in I/R injury, we utilized Factor B knockout mice in the intestinal model. Our results showed that Factor B knockout mice were not protected from local tissue injury, and their complement system was activated in the local tissues by nIgM during I/R. These results indicated that the lectin complement pathway operates immediately downstream of the nIgM-ischemic antigen interaction during intestinal I/R. Furthermore, the classical complement pathway also appears to interact with the of nIgM-ischemic antigen immunocomplex. Finally, the alternative complement pathway is not involved in I/R injury induction in the current intestinal model.

The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation.

Antigen (Ag) capture and presentation onto major histocompatibility complex (MHC) class II molecules by B lymphocytes is mediated by their surface Ag receptor (B cell receptor [BCR]). Therefore, the transport of vesicles that carry MHC class II and BCR-Ag complexes must be coordinated for them to converge for processing. In this study, we identify the actin-associated motor protein myosin II as being essential for this process. Myosin II is activated upon BCR engagement and associates with MHC class II-invariant chain complexes. Myosin II inhibition or depletion compromises the convergence and concentration of MHC class II and BCR-Ag complexes into lysosomes devoted to Ag processing. Accordingly, the formation of MHC class II-peptides and subsequent CD4 T cell activation are impaired in cells lacking myosin II activity. Therefore, myosin II emerges as a key motor protein in BCR-driven Ag processing and presentation.

Activation of the lectin pathway by natural IgM in a model of ischemia/reperfusion injury.

Reperfusion of ischemic tissues elicits an acute inflammatory response involving serum complement, which is activated by circulating natural IgM specific to self-Ags exposed by ischemia. Recent reports demonstrating a role for the lectin pathway raise a question regarding the initial events in complement activation. To dissect the individual roles of natural IgM and lectin in activation of complement, mice bearing genetic deficiency in early complement, IgM, or mannan-binding lectin were characterized in a mesenteric model of ischemia reperfusion injury. The results reveal that IgM binds initially to ischemic Ag providing a binding site for mannan-binding lectin which subsequently leads to activation of complement and injury.

Comparison of biochemical and immunochemical properties of myosin II in taeniid parasites.

Type II myosins are highly conserved proteins, though differences have been observed among organisms, mainly in the filamentous region. Myosin isoforms have been identified in Taenia solium, a helminth parasite of public health importance in many developing countries. These isoforms are probably associated with the physiological requirements of each developmental stage of the parasite. In this paper we extend the characterization of myosin to several other Taenia species. Type II myosins were purified from the larvae (cysticerci) of Taenia solium, T. taeniaeformis and T. crassiceps and the adult stages of T. solium, T. taeniaeformis and T. saginata. Rabbit polyclonal antibodies against some of these myosins were specific at high dilutions but cross-reacted at low dilutions. ATPase activity was evaluated and kinetic values were calculated for each myosin. Homologous actin-myosin interactions increased both the affinity of myosin for ATP and the hydrolysis rate. The results indicate immunological and biochemical differences among taeniid myosins. This variability suggests that different isoforms are found not only in different taeniid species but also at different developmental stages. Further characterization of myosin isoforms should include determination of their amino acid composition.

Muscular myosin isoforms of Taenia solium (Cestoda).

Type II myosin, the primary component of the thick filament of muscle fibers, is organized as a dimeric high molecular weight protein, and is composed of a pair of heavy chains (MHC) and two pairs of light chains. Myosin II transforms ATP energy into mechanical force. All type II myosins are conserved proteins but they have two variable regions that are located in different places of the molecule. Myosin molecules are encoded by a multigene family and many isoforms are generated. The expression of myosins depends on the developmental stage and on the type and degree of contractile activity and tissue, therefore several myosin isoforms are found in the same organism. Here we describe the use of different techniques that allowed demonstrating the presence of isoforms of the heavy chain type II myosin of Taenia solium cysticerci (larvae) and tapeworms (adults), a cestode parasite of importance in public health in many developing countries. Myosin was purified and used in comparative proteolytic fragmentation, ATPase activity, detection of antigenic differences and electrophoretic separation. The results obtained showed biochemical and immunochemical differences among cysticerci and tapeworms, and demonstrate the presence of myosin isoforms in T. solium that are probably associated to physiological requirements of each developmental stage.

Possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation in Ehrlich ascites tumor cells.

Osmotic shrinkage of Ehrlich ascites tumor cells (EATC) elicited translocation of myosin II from the cytosol to the cortical region, and swelling elicits concentration of myosin II in the Golgi region. Rho kinase and p38 both appeared to be involved in shrinkage-induced myosin II reorganization. In contrast, the previously reported shrinkage-induced actin polymerization [Pedersen et al. (1999) Exp. Cell Res. 252, 63-74] was independent of Rho kinase, p38, myosin light chain kinase (MLCK), and protein kinase C (PKC), which thus do not exert their effects on the shrinkage-activated transporters via effects on F-actin. The subsequent F-actin depolymerization, however, appeared MLCK- and PKC-dependent, and the initial swelling-induced F-actin depolymerization was MLCK-dependent; both effects were apparently secondary to kinase-mediated effects on cell volume changes. NHE1 in EATC is activated both by osmotic shrinkage and by the serine/threonine phosphatase inhibitor Calyculin A (CL-A). Both stimuli caused Rho kinase-dependent myosin II relocation to the cortical cytoplasm, but in contrast to the shrinkage-induced F-actin polymerization, CL-A treatment elicited a slight F-actin depolymerization. Moreover, Rho kinase inhibition did not significantly affect NHE1 activation, neither by shrinkage nor by CL-A. Implications for the possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation are discussed.