William A. Muller

Differentiation of Phagocytic Monocytes into Lymph Node Dendritic Cells In Vivo

We investigated the differentiation and trafficking of inflammatory monocytes that phagocytosed subcutaneously injected fluorescent microspheres. As expected, most of the monocytes became microsphere+ macrophages, which remained in subcutaneous tissue. However, about 25% of latex+ cells migrated to the T cell area of draining lymph nodes, where they expressed dendritic cell (DC)-restricted markers and high levels of costimulatory molecules. Microsphere-transporting cells were distinct from resident skin DCs, and this transport was reduced by more than 85% in monocyte-deficient osteopetrotic mice. Thus, a substantial minority of inflammatory monocytes carry phagocytosed particles to lymph nodes and differentiate into DCs.

The Abundant NK Cells in Human Secondary Lymphoid Tissues Require Activation to Express Killer Cell Ig-Like Receptors and Become Cytolytic

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56dimCD16+ and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56brightCD16− phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin− NK cell compartment in lymph nodes and related tissues probably outnumbers perforin+ NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.

The Leukotriene C4 Transporter MRP1 Regulates CCL19 (MIP-3β, ELC)–Dependent Mobilization of Dendritic Cells to Lymph Nodes

Adaptive immune responses begin after antigen-bearing dendritic cells (DCs) traffic from peripheral tissues to lymph nodes. Here, we show that DC migration from skin to lymph nodes utilizes the leukotriene C(4) (LTC(4)) transporter multidrug resistance-associated protein 1 (MRP1). DC mobilization from the epidermis and trafficking into lymphatic vessels was greatly reduced in MRP1(-/-) mice, but migration was restored by exogenous cysteinyl leukotrienes LTC(4) or LTD(4). In vitro, these cysteinyl leukotrienes promoted optimal chemotaxis to the chemokine CCL19, but not to other related chemokines. Antagonism of CCL19 in vivo prevented DC migration out of the epidermis. Thus, MRP-1 regulates DC migration to lymph nodes, apparently by transporting LTC(4), which in turn promotes chemotaxis to CCL19 and mobilization of DCs from the epidermis.

CD99 plays a major role in the migration of monocytes through endothelial junctions

CD99 is a heavily O-glycosylated 32-kD type I transmembrane protein that is expressed on most hematopoietic cells. We show here that CD99 is expressed on endothelial cells and is concentrated at the borders between confluent cells. We found that a monoclonal antibody to CD99, hec2, selectively inhibited diapedesis of monocytes across endothelial cells by >90%. Diapedesis involved the homophilic interaction of CD99 on monocytes with CD99 on endothelial junctions. CD99 functioned distally to the point at which platelet-endothelial cell adhesion molecule 1 (PECAM-1, also known as CD31), another adhesion molecule involved in transmigration, played its critical role. Confocal microscopy showed that anti–PECAM-1 arrested leukocytes on the apical surface of endothelium, whereas blocking CD99 arrested monocytes at a point where they were partially through the junction. Therefore, diapedesis, the forward migration of leukocytes through endothelial junctions, is regulated sequentially by two distinct molecules, PECAM-1 and CD99.

Locomotion of monocytes on endothelium is a critical step during extravasation

Monocytes, like all leukocytes, undergo a series of sequential steps during extravasation from blood into tissues: tethering, rolling, adhesion and diapedesis. We have discovered an essential step, which we call locomotion, in which the monocyte moves from a site of firm adhesion to the nearest junction to begin diapedesis. Blocking CD11a-CD18 and CD11b-CD18 on human monocytes or adhesion molecules ICAM-1 and ICAM-2 on endothelial cells prevented the monocytes from reaching junctions. The blocked monocytes spun in circles as if they were unable to direct their movement despite being able to adhere and polarize normally. This step fills a gap in the paradigm of extravasation as a multistep process.

Targeted recycling of PECAM from endothelial surface-connected compartments during diapedesis

Leukocytes enter sites of inflammation by squeezing through the borders between endothelial cells that line postcapillary venules at that site. This rapid process, called transendothelial migration (TEM) or diapedesis, is completed within 90 s after a leukocyte arrests on the endothelial surface. In this time, the leukocyte moves in ameboid fashion across the endothelial borders, which remain tightly apposed to it during transit. It is not known how the endothelial cell changes its borders rapidly and reversibly to accommodate the migrating leukocyte. Here we show that there is a membrane network just below the plasmalemma at the cell borders that is connected at intervals to the junctional surface. PECAM-1, an integral membrane protein with an essential role in TEM, is found in this compartment and constitutively recycles evenly along endothelial cell borders. During TEM, however, recycling PECAM is targeted to segments of the junction across which monocytes are in the act of migration. In addition, blockade of TEM with antibodies against PECAM specifically blocks the recruitment of this membrane to the zones of leukocyte migration, without affecting the constitutive membrane trafficking.

The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling.

Vascular endothelial growth factor (VEGF) receptor Flk-1/KDR in endothelial cells is activated during vasculogenesis and angiogenesis upon ligand-receptor interaction. Activated Flk-1/KDR has been shown to recruit Src homology 2 domain-containing signaling molecules that are known to serve as links to the activation of the mitogen-activated protein (MAP) kinase signaling pathway. To define the functional significance of phosphatidylinositol (PI) 3-kinase in VEGF signaling, we have examined its role in human umbilical vein endothelial cell (HUVEC) cycle progression. We show herein that p85, the regulatory subunit of PI 3-kinase, is constitutively associated with Flk-1/KDR. The treatment of HUVECs with VEGF promoted tyrosine autophosphorylation of Flk-1/KDR and also induced phosphorylation of p85. This was followed by an increase in the PI 3-kinase activity, which was sensitive to wortmannin, a potent PI 3-kinase inhibitor. VEGF also induced a striking activation of MAP kinase in a time-dependent manner. Inhibition studies with both a dominant-negative p85 mutant and the PI 3-kinase inhibitor, wortmannin, were employed to show for the first time that VEGF-stimulated PI 3-kinase modulates MAP kinase activation and nuclear events such as transcription from c-fos promoter and entry into the synthesis (S)-phase. Our data demonstrate the importance of PI 3-kinase as a necessary signaling component of VEGF-mediated cell cycle progression.

Leukocyte-endothelial cell interactions in the inflammatory response.

I nflammation is the response of vascularized tissue to damage. It is a complex response set in motion in any tissue by virtually any injury due to any cause. It is an attempt to restore homeostasis. In the early history of Homo sapiens, when lifespans were relatively short and our ancestors were likely to die from infectious diseases and trauma, the ability to fight off pathogens and heal wounds was even more critical for the survival of the species than it is today. The inflammatory response is still critical for these purposes. However, now that we are living longer, we have come to realize that inflammation is a doubleedged sword. The same inflammatory processes that are so effective at clearing pathogens and repairing damaged skin are the cause of most human pathology today. Atherosclerosis, autoimmune diseases, pulmonary fibrosis, asthma, and many other common human afflictions are all conditions that result when the inflammatory response is uncontrolled, self-directed, or in the wrong place at the wrong time. These conditions generally affect humans past the traditional breeding age and thus these untoward aspects of inflammation were not selected out during evolution. As a result, there is now far more effort to develop potent anti-inflammatory therapies than there is to develop treatments to boost the inflammatory response. What is needed is the therapeutic equivalent of “smart bombs”—the ability to selectively inhibit inflammation where it is damaging without interfering with beneficial functions of the inflammatory response. To design rational therapies, a more thorough understanding of the cellular and molecular basis of inflammation is necessary. A great deal of research has been carried out in this area over the past two decades and significant progress has been made. Along with this progress has come an array of jargon and hundreds of molecules denoted by cluster of differentiation (CD) numbers that make following the field daunting for those not involved in it (see Table 1) This brief overview is intended to introduce the nonspecialist to the general concepts in the field and the important areas for future research. A general paradigm of leukocyte-endothelial cell interactions in inflammation is presented that is generally agreed on in the field. This is obviously oversimplified; alternative interactions do exist. In particular, there are certain adhesion molecules that are critical to inflammation in some tissues but not in others. The reader is referred to more detailed reviews cited in each section for a more thorough discussion.

The contribution of monocyte infection and trafficking to viral persistence, and maintenance of the viral reservoir in HIV infection

Cellular viral reservoirs and anatomic sanctuary sites allow continuing HIV‐1 replication in patients with suppressed plasma viremia who are receiving highly active antiretroviral therapy and prevent eradication of HIV‐1 by these regimens. Cells of macrophage lineage, including monocytes subsets within the blood, play a role in HIV‐1 persistence. Evidence of sequence evolution in blood monocytes, in comparison to resting CD4+ T cells, demonstrates their distinct contribution to plasma viremia. There is evidence to suggest that a specific monocyte subset, of CD14loCD16hi phenotype, is more susceptible to HIV‐1 infection than the majority of blood monocytes. Trafficking of monocytes through various tissues following their emigration from the bloodstream allows these cells to differentiate into tissue macrophages, or potentially to egress from the tissues as migratory dendritic cells. This review provides an evaluation of the contribution of monocytes to HIV‐1 persistence and the HIV‐1 reservoir, essential for the effective design of therapeutic eradication strategies.

Mechanisms of Transendothelial Migration of Leukocytes

A great deal of progress has been made recently in understanding the molecules and mechanisms that regulate transendothelial migration of leukocytes, or diapedesis, a critical step in the inflammatory response. This review focuses mainly on the active role of the endothelial cell in this process as it occurs at endothelial cell borders. It discusses some of the many molecules that have been reported to play a role in transendothelial migration and asks why so many molecules seem to be involved. The concept is emerging that diapedesis itself can be dissected into sequential steps controlled by specific molecule(s) at the endothelial cell border. Several mechanisms have been shown to play a critical role in transendothelial migration including signals derived from clustering of apically disposed intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, disruption or loosening of adherens junctions, and targeted recycling of platelet/endothelial cell adhesion molecule and other molecules from the recently described lateral border recycling compartment. A hypothesis that integrates the various known mechanisms is proposed.