Martin J. Brown

Stat4 Is Expressed in Activated Peripheral Blood Monocytes, Dendritic Cells, and Macrophages at Sites of Th1-Mediated Inflammation

Stat4 is a key transcription factor involved in promoting cell-mediated immunity, whose expression in mature cells has been reported to be restricted to T and NK cells. We demonstrate here, however, that Stat4 expression is not restricted to lymphoid cells. In their basal state, monocytes do not express Stat4. Upon activation, however, IFN-γ- and LPS-treated monocytes and dendritic cells express high levels of Stat4. Monocyte-expressed Stat4 in humans is phosphorylated in response to IFN-α, but not IL-12. In contrast, the Th2 cytokines, IL-4 and IL-10, specifically down-regulate Stat4 expression in activated monocytes, while having little effect on Stat6 expression. Moreover, macrophages in synovial tissue obtained from patients with rheumatoid arthritis express Stat4 in vivo, suggesting a potential role in a prototypical Th1-mediated human disease. IFN-α-induced Stat4 activation in human monocytes represents a previously unrecognized signaling pathway at sites of Th1 inflammation.

Rigidity of Circulating Lymphocytes Is Primarily Conferred by Vimentin Intermediate Filaments

Lymphocytes need rigidity while in circulation, but must abruptly become deformable to undergo transmigration into tissue. Previously, the control of leukocyte deformability has been attributed to microfilaments or microtubules, but the present studies demonstrate the greater importance of vimentin intermediate filaments (IFs). In circulating T lymphocytes, IFs form a distinctive spherical cage that undergoes a rapid condensation into a juxtanuclear aggregate during chemokine-induced polarization. Measurements of the resistance of peripheral blood T lymphocytes to global deformation demonstrate that their rigidity is primarily dependent on intact vimentin filaments. Microtubules, in contrast, are not sufficient to maintain rigidity. Thus, vimentin IFs are a primary source of structural support in circulating human lymphocytes, and their regulated collapse is likely to be an essential element in chemokine-induced transendothelial migration.

Protein kinase C θ is expressed in mast cells and is functionally involved in Fcε receptor I signaling

We investigated possible expression and function in mast cells ofprotein kinase C (PKC) θ, a member of the PKC family withdemonstrated function in a limited range of cell types. We found thatPKC θ is expressed in bone marrow‐derived mast cells and in the RBL‐2H3 mast cell line. PKC θ underwent translocation to the membranein response to Fcɛ receptor I (FcɛR I) activation. Receptoractivation induced phosphorylation of PKC θ. The tyrosinephosphorylation of PKC θ is delayed relative to PKC δ and coincidestemporally with PKC θ association with c‐src family members Lyn andSrc. Studies of RBL‐2H3 cells transduced with PKC θ constructsindicated a role for PKC θ in receptor‐induced activation ofextracellular regulated kinases, interleukin‐3 gene transcription, anddegranulation in response to antigen stimulation. These studies extendthe known functions of PKCθ to another important immune cell type andindicate the concurrent participation of multiple PKCs in the FcɛRI‐mediated response of mast cells.

Cutting Edge: Integration of Human T Lymphocyte Cytoskeleton by the Cytolinker Plectin

Chemokine-induced polarization of lymphocytes involves the rapid collapse of vimentin intermediate filaments (IFs) into an aggregate within the uropod. Little is known about the interactions of lymphocyte vimentin with other cytoskeletal elements. We demonstrate that human peripheral blood T lymphocytes express plectin, an IF-binding, cytoskeletal cross-linking protein. Plectin associates with a complex of structural proteins including vimentin, actin, fodrin, moesin, and lamin B in resting peripheral blood T lymphocytes. During chemokine-induced polarization, plectin redistributes to the uropod associated with vimentin and fodrin; their spatial distribution indicates that this vimentin-plectin-fodrin complex provides a continuous linkage from the nucleus (lamin B) to the cortical cytoskeleton. Overexpression of the plectin IF-binding domain in the T cell line Jurkat induces the perinuclear aggregation of vimentin IFs. Plectin is therefore likely to serve as an important organizer of the lymphocyte cytoskeleton and may regulate changes of lymphocyte cytoarchitecture during polarization and extravasation.

Assessment of lymphocyte-mediated cytotoxicity using flow cytometry.

Cytotoxic lymphocytes, including cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill target cells by releasing granules containing perforin and granzymes, and/or via Fas-Fas ligand interactions. Both pathways lead to prompt activation within target cells of caspase cascades responsible for apoptosis induction and cell death. We have utilized cell-permeable fluorogenic caspase substrates and multiparameter flow cytometry to detect caspase activation in target cells, and applied these tools to quantify and visualize cytotoxic lymphocyte activities. This novel assay, referred to as the flow cytometric cytotoxicity (FCC) assay, is a nonradioactive single-cell-based assay that provides a more rapid, biologically informative, and sensitive approach to measure cytotoxic lymphocyte activity when compared to other assays such as the 51chromium (51Cr) release assay. In addition, the FCC assay can be used to study CTL-mediated killing of primary target cells of different cell lineages that are frequently not amenable to study by the 51Cr release assay. Furthermore, the FCC assay enables evaluation of the phenotype and fate of both target and effector cells, and as such, provides a useful new approach to illuminate the biology of cytotoxic lymphocytes.

Polyethylene glycol-mediated infection of non-permissive mammalian cells with semliki forest virus: application to signal transduction studies

Semliki Forest Virus (SFV) vectors allow the subcloning of a gene of interest directly in the expression vector, thus avoiding the need to select and purify viral recombinants, making this viral expression system attractive over many others for mammalian protein expression. We now describe a novel and generally applicable method for infection of non-permissive mammalian cells with SFV, that greatly enhances the utility of this expression system. We demonstrate that the hygroscopic polymer poly (ethylene glycol), PEG, promotes the infectivity of cells by SFV under conditions that did not promote cell-cell fusion. We also found that the PEG-induced infection and expression of an exogenous protein (green fluorescent protein, GFP) did not elevate the basal tyrosine kinase activity, induce a stress-activated responses, or result in aberrant cell responses. Expression of GFP tagged-Vav, an activator of stress-activated protein kinase (SAPK/JNK), resulted in the expected induction of JNK activity and in the normal redistribution of Vav in response to engagement of the high affinity receptor for IgE (FcepsilonRI). Thus, our findings that PEG allows the infection of non-permissive cells by SFV makes this system extremely attractive for expression of proteins in mammalian cells, and studies on signal transduction and cellular localization in immune and non-immune cells.

T-cell activation: Interplay at the interface

Recent studies have shown that, when a T cell interacts with a cognate antigen-presenting cell, an organized adhesive contact is formed between the two cells by a process which involves the dynamic, three-dimensional redistribution of entire signaling assemblies.