Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines

Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles—the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation—with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature

The Many Roles of CD40 in Cell-Mediated Inflammatory Responses

In addition to B cells, CD40 is expressed by a variety of cells including dendritic cells, monocytes, fibroblasts and vascular endothelial cells. As discussed here by Robert Stout and Jill Suttles, CD40-CD40 ligand interactions can influence many aspects of T-cell-mediated inflammatory responses, such as cell extravasation, production of inflammatory and chemotactic cytokines, as well as activation of macrophage effector functions

T Cells Bearing the CD44\u3csup\u3eHi\u3c/sup\u3e Memory Phenotype Display Characteristics of Activated Cells in G1 Stage of Cell Cycle

T cells capable of anamnestic proliferative responses to antigen in vitro (i.e., memory cells) have been shown to display the CD44hi CD45RBlo surface phenotype. To assess the state of activation of these T cells, CD4+ T cells expressing the CD44hi or CD45RBlo phenotype were compared to CD4+ T cells expressing the CD44lo or CD45RBhi phenotype in the context of expression of the activated (asiata-GhiM1) vs resting (asialo-GloM1) phenotype and in the context of cell size, total protein content, and total RNA content. Dual fluorescence analysis demonstrated that all CD4+ T cells expressing the CD44hi phenotype also expressed the asialo-GhiM1 phenotype associated with cell activation. In vitro proliferative assays confirmed that the CD4+ asialo-GhiM1, the CD4+ CD45RBlo, and the CD4+ CD44hi FACS-sorted populations displayed stronger in vitro responsiveness to stimulation with immobilized anti-CD3 mAB than the CD4+ asialo-GloM1, CD45RBhi, or CD44lo populations. Acridine orange analysis of sorted CD44hi/lo fractions revealed that the diploid (G1) population of the CD44hi T cells displayed a higher mean RNA content than the CD44lo T cells. Similarly, the CD44hi T cells displayed a higher mean cell size and a higher mean total protein content than the CD44lo CD4+ T cells. Similar results were obtained with asialo-GM1 and CD45RB subsets of CD4+ T cells. The basal rate of protein synthesis, as determined by [3H]leucine incorporation, was ≈50% higher in the CD44hi small CD4+ T cells than in the CD44lo CD4+ T cells. Based on the knowledge that cell size, total protein and RNA content, and responsiveness to signals inducing proliferation are lowest in GO stage of cycle and increase through Gl stage of cycle, it appears that the CD44hi CD45RBlo T cells exist in a higher activation state than CD44lo CD45RBhi T cells. The previously demonstrated association of CD44hi CD45RBlo phenotype with memory T cells suggests that the CD44hi memory T cells are maintained in G1 (not necessarily cycling) rather than resting out of cycle in G0

Evidence for Involvement of TNF-α in the Induction Phase and IFN-β in the Effector Phase of Antiproliferative Activity of Splenic Macrophages

Splenic macrophages play a key role in regulating cell proliferation during a variety of chronic perturbations of the hematopoietic system. This regulatory activity is in sharp contrast to the activities of inflammatory monocytes/macrophages in that it is not dominated by the secretion of prostaglandins or toxic metabolites such as peroxides. A productive model for studying these nontoxic regulatory activities of splenic macrophages has been provided by macrophages generated in vitro (Mø-c) during autologous spleen cell culture. The Mø-c effectively inhibit (\u3e90%) lymphocyte proliferation by inhibiting G1→S phase progression without inhibiting the production of interleukins by the lymphocytes. Conditioned medium from Mø-c activated with LPS + rIFN-γ effected a similar G1 arrest of activated lymphocytes. The involvement of IFN-β in effecting the antiproliferative activity is suggested by (1) the ability of monospecific anti-IFN-β mAB, but not anti-TGF-β, anti-IL-1, anti-TNF-α, or anti-IFN-γ, to neutralize the antiproliferative activity in the Mø-c supernatants and (2) the ability of purified IFN-β to effect a similar inhibition of cell proliferation (i.e., G1 arrest without inhibition of interleukin production). rTNF-α and rIFN-γ could not effect such an inhibition of cell proliferation and did not synergize with IFN-β in producing such an antiproliferative effect. The Mø-c could be activated to effector function by a combination of LPS + rIFN-γ or rTNF-α + rIFN-γ, but not by any one of those reagents alone. LPS alone was sufficient to stimulate TNF-α production by the Mø-c. Activation of the Mø)-c by LPS + rIFN-γ could be completely blocked by anti-TNF-a antibodies. These data suggest that the Mø-c can be induced to produce inhibitory levels of cytostatic cytokines by a TNF-α autocrine loop that is IFN-γ dependent. The in vivo relevance of this effector mechanism is suggested by, and discussed in the context of, the recent reports of spontaneous production of IFN-β during immunological disorders

Functional Characteristics of in Vitro Generated Macrophages: A Transient Refractory State Precedes Reinducibility of a Spatially Restricted, Possibly Contact-Dependent, Cytostatic Mechanism

Adherent layers of macrophages (Mφ-c) generated in vitro from splenic precursors previously have been shown to inhibit proliferation of normal and neoplastic cells by mechanisms that do not involve the secretion of significant quantities of prostaglandins, peroxides, or proteases. In the current report, it is demonstrated that the effective range of the cytostatic effect of adherent Mφ-c is so low that cytostasis can be circumvented by preventing the target cells from settling to the adherent layer by incubating the cultures of targets and Mφ-c on a rocking platform. Despite the paucity of production of inhibitory mediators, the Mφ-c do produce sufficient IL-1 to restore IL-2 production capacity to macrophage-depleted lymphocyte populations. The cytostatic activity of the Mφ-c is an inducible event and remains for several days in the continuous presence of mitogen-activated lymphocytes or LPS + lymphokines. Once the activators are removed the cytostatic activity rapidly declines, reaching background levels in 24-48 hr. Maximal cytostatic activity can be reinduced but only 48 hr or longer after the primary activators were removed. A transient period in which the Mφ-c are refractory to reactivation can be demonstrated to exist for 1-2 days after removal of the primary activators

T cell signaling of macrophage function in inflammatory disease. Front Biosci

Macrophages play diverse roles in episodic T cell-mediated inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, function as accessory cells for T cell activation, as pro-inflammatory cells, as effector cells which mediate tissue damage, and as anti-inflammatory cells which promote wound healing. In addition to the many roles of T cell-derived cytokines in differentially modulating these diverse macrophage activities, research over the last few years has demonstrated that contact-dependent signaling which occurs during T cell-macrophage adhesion is a critical triggering event in the activation of macrophage function. Substantial research emphasis has been placed on CD40 as a mediator of contact dependent signaling. However, other membrane-anchored receptor:ligand pairs may also contribute to the stimulation of macrophage function. This is a brief review of the rapidly expanding, but still incomplete, knowledge of how T cells, through both contact-dependent and cytokine signals, regulate macrophage function during inflammatory disease

Panning T Cells on Vascular Endothelial Cell Monolayers: A Rapid Method for Enriching Naive T Cells

A key functional/phenotypic difference between naive and memory T cells is the ability of memory and activated T cells to home to sites of inflammation by adhering to vascular endothelial cells. To determine if this trait could be used to separate naive T cells from memory T cells. CD4+ T cells were incubated with monolayers of IFN-γ-primed vascular endothelial cells after which the phenotypic and functional characteristics of the nonadherent population were assayed. The nonadherent population 1) contained a five-fold decrease in the frequency of cells displaying the CD44high/CD45RBlow memory phenotype and 2) responded well to allostimulation but displayed a reduced ability to respond to immobilized anti-CD3 antibody and, when isolated from ovalbumin-immunized mice, displayed a reduced recall response to ovalbumin in vitro. These studies demonstrate that two brief incubations of T cells with monolayers of IFN-γ-primed endothelial cells can significantly enrich for naive T cells as determined by both phenotypic and functional analyses