Varsha Kumar

Cell-derived anaphylatoxins as key mediators of antibody-dependent type II autoimmunity in mice

Complement C5a, a potent anaphylatoxin, is a candidate target molecule for the treatment of inflammatory diseases, such as myocardial ischemia/reperfusion injury, RA, and the antiphospholipid syndrome. In contrast, up until now, no specific contribution of C5a and its receptor, C5aR, was recognized in diseases of antibody-dependent type II autoimmunity. Here we identify C5a as a novel key mediator of autoimmune hemolytic anemia (AIHA) and show that mice lacking C5aR are partially resistant to this IgG autoantibody-induced disease model. Upon administration of anti-erythrocyte antibodies, upregulation of activating Fcgamma receptors (FcgammaRs) on Kupffer cells, as observed in WT mice, was absent in C5aR-deficient mice, and FcgammaR-mediated in vivo erythrophagocytosis was impaired. Surprisingly, in mice deficient in FcgammaRI and FcgammaRIII, anti-erythrocyte antibody-induced C5 and C5a production was abolished, demonstrating the existence of a previously unidentified FcgammaR-mediated C5a-generating pathway. These results show that the development of a full-blown antibody-dependent autoimmune disease requires C5a--produced by and acting on FcgammaR--and may suggest therapeutic benefits of C5 and/or C5a/C5aR blockade in AIHA and other diseases closely related to type II autoimmune injury.

Macrophages Induce the Inflammatory Response in the Pulmonary Arthus Reaction through Gαi2 Activation That Controls C5aR and Fc Receptor Cooperation

Complement and FcγR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcγRIIB, increased levels of activating FcγRIII, and highly induced FcγR-mediated TNF-α and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of Gi-type G protein signaling in C5aR-mediated control of the regulatory FcγR system in vitro, and analysis of the various C5aR-, FcγR-, and Gi-deficient mice verifies the importance of Gαi2-associated C5aR and the FcγRIII-FcγRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcγRIII-positive cells into C5aR- and FcγRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcγRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcγRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Gαi2-dependent signal for modulating the two opposing FcγR, FcγRIIB and FcγRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.

Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway

Adaptive immune responses are characterized by substantial restructuring of secondary lymphoid organs. The molecular and cellular factors responsible for virus-induced lymphoid remodeling are not well known to date. Here we applied optical projection tomography, a mesoscopic imaging technique, for a global analysis of the entire 3-dimensional structure of mouse peripheral lymph nodes (PLNs), focusing on B-cell areas and high endothelial venule (HEV) networks. Structural homeostasis of PLNs was characterized by a strict correlation between total PLN volume, B-cell volume, B-cell follicle number, and HEV length. After infection with lymphocytic choriomeningitis virus, we observed a substantial, lymphotoxin (LT) beta-receptor-dependent reorganization of the PLN microarchitecture, in which an initial B-cell influx was followed by 3-fold increases in PLN volume and HEV network length on day 8 after infection. Adoptive transfer experiments revealed that virus-induced PLN and HEV network remodeling required LTalpha(1)beta(2)-expressing B cells, whereas the inhibition of vascular endothelial growth factor-A signaling pathways had no significant effect on PLN expansion. In summary, lymphocytic choriomeningitis virus-induced PLN growth depends on a vascular endothelial growth factor-A-independent, LT- and B cell-dependent morphogenic pathway, as revealed by an in-depth mesoscopic analysis of the global PLN structure.

Coordinated Regulation of Lymph Node Vascular–Stromal Growth First by CD11c+ Cells and Then by T and B Cells

Lymph node blood vessels play important roles in the support and trafficking of immune cells. The blood vasculature is a component of the vascular–stromal compartment that also includes the lymphatic vasculature and fibroblastic reticular cells (FRCs). During immune responses as lymph nodes swell, the blood vasculature undergoes a rapid proliferative growth that is initially dependent on CD11c+ cells and vascular endothelial growth factor (VEGF) but is independent of lymphocytes. The lymphatic vasculature grows with similar kinetics and VEGF dependence, suggesting coregulation of blood and lymphatic vascular growth, but lymphatic growth has been shown to be B cell dependent. In this article, we show that blood vascular, lymphatic, and FRC growth are coordinately regulated and identify two distinct phases of vascular–stromal growth—an initiation phase, characterized by upregulated vascular–stromal proliferation, and a subsequent expansion phase. The initiation phase is CD11c+ cell dependent and T/B cell independent, whereas the expansion phase is dependent on B and T cells together. Using CCR7−/− mice and selective depletion of migratory skin dendritic cells, we show that endogenous skin-derived dendritic cells are not important during the initiation phase and uncover a modest regulatory role for CCR7. Finally, we show that FRC VEGF expression is upregulated during initiation and that dendritic cells can stimulate increased fibroblastic VEGF, suggesting the scenario that lymph node-resident CD11c+ cells orchestrate the initiation of blood and lymphatic vascular growth in part by stimulating FRCs to upregulate VEGF. These results illustrate how the lymph node microenvironment is shaped by the cells it supports.

Distinct molecular composition of blood and lymphatic vascular endothelial cell junctions establishes specific functional barriers within the peripheral lymph node.

Lymph nodes are strategically localized at the interfaces between the blood and lymphatic vascular system, delivering immune cells and antigens to the lymph node. As cellular junctions of endothelial cells actively regulate vascular permeability and cell traffic, we have investigated their molecular composition by performing an extensive immunofluorescence study for adherens and tight junction molecules, including vascular endothelium (VE)‐cadherin, the vascular claudins 1, 3, 5 and 12, occludin, members of the junctional adhesion molecule family plus endothelial cell‐selective adhesion molecule (ESAM)‐1, platelet endothelial cell adhesion molecule‐1, ZO‐1 and ZO‐2. We found that junctions of high endothelial venules (HEV), which serve as entry site for naive lymphocytes, are unique due to their lack of the endothelial cell‐specific claudin‐5. LYVE‐1+ sinus‐lining endothelial cells form a diffusion barrier for soluble molecules that arrive at the afferent lymph and use claudin‐5 and ESAM‐1 to establish characteristic tight junctions. Analysis of the spatial relationship between the different vascular compartments revealed that HEV extend beyond the paracortex into the medullary sinuses, where they are protected from direct contact with the lymph by sinus‐lining endothelial cells. The specific molecular architecture of cellular junctions present in blood and lymphatic vessel endothelium in peripheral lymph nodes establishes distinct barriers controlling the distribution of antigens and immune cells within this tissue.

Dual regulation of IRF4 function in T and B cells is required for the coordination of T–B cell interactions and the prevention of autoimmunity

Female mice lacking DEF6 and SWAP70 develop a lupus-like syndrome through dysregulation of IRF4 in activated B cells and plasma cells.

In vivo analysis of uropod function during physiological T cell trafficking.

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonactivated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine-coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod contraction during lymphocyte recirculation.

A Dendritic-Cell-Stromal Axis Maintains Immune Responses in Lymph Nodes

Within secondary lymphoid tissues, stromal reticular cells support lymphocyte function, and targeting reticular cells is a potential strategy for controlling pathogenic lymphocytes in disease. However, the mechanisms that regulate reticular cell function are not well understood. Here we found that during an immune response in lymph nodes, dendritic cells (DCs) maintain reticular cell survival in multiple compartments. DC-derived lymphotoxin beta receptor (LTβR) ligands were critical mediators, and LTβR signaling on reticular cells mediated cell survival by modulating podoplanin (PDPN). PDPN modulated integrin-mediated cell adhesion, which maintained cell survival. This DC-stromal axis maintained lymphocyte survival and the ongoing immune response. Our findings provide insight into the functions of DCs, LTβR, and PDPN and delineate a DC-stromal axis that can potentially be targeted in autoimmune or lymphoproliferative diseases.

Optical projection tomography reveals dynamics of HEV growth after immunization with protein plus CFA and features shared with HEVs in acute autoinflammatory lymphadenopathy

The vascular–stromal compartment of lymph nodes is important for lymph node function, and high endothelial venules (HEVs) play a critical role in controlling the entry of recirculating lymphocytes. In autoimmune and autoinflammatory diseases, lymph node swelling is often accompanied by apparent HEV expansion and, potentially, targeting HEV expansion could be used therapeutically to limit autoimmunity. In previous studies using mostly flow cytometry analysis, we defined three differentially regulated phases of lymph node vascular–stromal growth: initiation, expansion, and the re-establishment of vascular quiescence and stabilization. In this study, we use optical projection tomography to better understand the morphologic aspects of HEV growth upon immunization with ovalbumin/CFA (OVA/CFA). We find HEV elongation as well as modest arborization during the initiation phase, increased arborization during the expansion phase, and, finally, vessel narrowing during the re-establishment of vascular quiescence and stabilization. We also examine acutely enlarged autoinflammatory lymph nodes induced by regulatory T cell depletion and show that HEVs are expanded and morphologically similar to the expanded HEVs in OVA/CFA-stimulated lymph nodes. These results reinforce the idea of differentially regulated, distinct phases of vascular–stromal growth after immunization and suggest that insights gained from studying immunization-induced lymph node vascular growth may help to understand how the lymph node vascular–stromal compartment could be therapeutically targeted in autoimmune and autoinflammatory diseases.

Automated Recovery of the Center of Rotation in Optical Projection Tomography in the Presence of Scattering

Finding the center of rotation is an essential step for accurate 3-D reconstruction in optical projection tomography. Unfortunately, current methods are not convenient since they require either prior scanning of a reference phantom, small structures of high intensity existing in the specimen, or active participation during the centering procedure. To solve these problems this paper proposes a fast and automatic center of rotation search method making use of parallel programming in graphics processing units. Our method is based on a two step search approach making use only of those sections of the image with high signal-to-noise ratio. We have tested this method both in nonscattering ex vivo samples and in in vivo specimens with a considerable contribution of scattering such as Drosophila melanogaster pupae, recovering in all cases the center of rotation with a precision 1/4 pixel or less.