Macrophage Precursors Research Articles

Expression profilings of 39 genes selected by ANOVA could separate precursors of murine dendritic cells and macrophages

Dendritic cells (DCs) and macrophages share some stages in the development and function of antigen presentation. But it is difficult to separate them from their precursors. We used one-way ANOVA (analysis of variances) on murine expression profilings of several hematopoietic cells associated with DCs and macrophages to find the genes with great differences across the cell groups. These groups were the DCs from spleen, cultivated DCs, DC precursors, DC progenitors, DC progenitor cell lines, hematopoietic stem cell (HSC), and bone marrow-derived macrophages. The data of expression profilings were all downloaded from GEO and ArrayExpress database. After the normalization of 11 housekeeping genes across 42 arrays, we got 39 genes (44 probesets) by analysis of one-way ANOVA (Bonferroni step-down) with p values cutoff of 0.05. These genes (probesets) could separate the hematopoietic cells well by the methods of unsupervised hierarchical clustering and principal component analysis (PCA). The class prediction also indicated that these genes could separate the precursors of DC and macrophages with 20 arrays composed of 5 cell types with the same normalization. The accuracy rate of class prediction was 90% (18/20). The genes selected by one-way ANOVA included those of MHC (major histocompatibility complex) and defense of immunity, cell adhesion, chemokine or its receptors, and transcription factors. The results indicated that these 39 genes could separate precursors of DC and macrophages very clearly. It was suggested that these genes might represent some important molecules that related with the precursors of DCs and macrophages, and were worthy for further study.

Immature monocytes acquire antigens from other cells in the bone marrow and present them to T cells after maturing in the periphery

Monocytes are circulating precursors for tissue macrophages and dendritic cells (DCs) but are not recognized to directly participate in antigen presentation. We developed techniques to label mouse monocyte subsets with particulate tracers in vivo. Gr-1lo but not Gr-1hi monocytes were stably labeled by intravenous injection of 0.5-μm microspheres. Gr-1hi monocytes could be labeled when the microspheres were injected after systemic depletion of blood monocytes and spleen macrophages. In this condition, the phagocytic tracer was transferred to immature bone marrow monocytes by neutrophils and B cells that first carried the particles to the bone marrow. Moreover, antigens from B cells or proteins conjugated to the tracer particles were processed for presentation by monocytes and could induce T cell responses in the periphery. Cell-associated antigen taken up by bone marrow monocytes was retained intracellularly for presentation of the antigen days later when monocyte-derived DCs migrated to lymph nodes or in vitro after differentiation with granulocyte/macrophage colony-stimulating factor. These data reveal that immature monocytes unexpectedly sample antigen from the bone marrow environment and that they can present these antigens after they leave the bone marrow.

Therapeutic approaches in bone pathogeneses: targeting the IKK/NF-κB axis

Bone erosion is a major hallmark of rheumatoid arthritis and is executed solely by the bone-resorbing cell, the osteoclast. This cell arises from macrophage precursors and differentiates into the mature polykaryon after stimulation with the receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor. Osteoclasts are recruited to sites of inflammation, or differentiate at these sites owing to elevated levels of circulating RANKL and other inflammatory cytokines secreted by cells in the inflamed tissue. Recent therapies to combat inflammatory bone erosion have focused on proximal and intracellular signaling molecules to attenuate osteoclast formation and activity. In this review, osteoclast differentiation, activation mechanisms, the role of the NF-κB pathway in inflammatory osteolysis and the relevant intervention approaches are presented briefly. The emphasis of this review will be on the RANKL–RANK–IκB kinase–NF-κB pathway and related antiosteolytic and anti-inflammatory modalities.

CLONING AND CHARACTERIZATION OF OSTEOCLAST PRECURSORS FROM THE RAW264.7 CELL LINE

Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-kappaB ligand (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell cultures, which are poorly suited to molecular and transgene studies because of the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study, we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP-positive multinuclear cells. Clones capable of forming large TRAP-positive multinuclear cells also expressed beta3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-kappaB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation.

Differential Requirement for Cell Division in the Development of Terminally Differentiated Neutrophils and Macrophages from Their Common Progenitors.

Neutrophils and monocytes are the main population of peripheral blood nucleated cells and progenies of common progenitor cells, CFU-GM, that are derived from hematopoietic stem cells. Monocytes are non-dividing immediate precursors for macrophages. Common clinical observation that monocytes more rapidly recover than do neutrophils in myelosuppressive patients indicates the difference in regulatory mechanism of terminal differentiation between neutrophils and macrophages. In this study, we investigated the cell division profiles in the differentiation pathway from common progenitors for neutrophils and macrophages to respective post-mitotic cells in culture. We prepared lineage-negative (Lin−) bone marrow cells from normal C57BL/6 mice by using immunomagnetic beads. The Lin− cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), a cytoplasmic dye that is equally diluted between daughter cells, and stained with anti-Sca-1 antibody and lineage antibodies. CFSEhighSca-1+Lin− cells were sorted on a FACSVantage or FACSAria. When the CFSEhighSca-1+Lin− cells were cultured in the presence of steel factor (SF) and IL-11 for 5 days, the resulting cells could be separated into four populations based on staining with anti-fms and anti-Gr-1 antibodies: fms-Gr-1−, fms+Gr-1−, fms−Gr-1+, and fms+Gr-1+ cells. The fms+Gr-1+ cells showed morphological features typical of macrophages. The commitment of fms−Gr-1+ cells into neutrophilic lineage was evident, although they exhibited an immature morphology. CFSE labeling pattern in fms+Gr-1+ macrophages was equal to that of fms−Gr-1+ neutrophilic cells, indicating that fms+Gr-1+ macrophages and fms−Gr-1+ neutrophilic cells passed through similar successive rounds of cell division in culture of CFSEhighSca-1+Lin− cells. These fms+Gr-1+ macrophages and fms−Gr-1+ neutrophilic cells were subsequently cultured for additional 3 days in the presence of SF, IL-11, IL-3, GM-CSF, G-CSF, M-CSF, erythropoietin, and thrombopoietin. While a decline in the number of fms+Gr-1+ macrophages was observed, the number of fms−Gr-1+ neutrophilic cells increased approximately 5-fold during subsequent culture. The proliferation of fms−Gr-1+ neutrophilic cells and no cell division of fms+Gr-1+ macrophages were confirmed by Ki-67 antibody staining, BrdU incorporation, and DNA staining. The cultured fms−Gr-1+ neutrophilic cells also predominantly acquired a mature morphology. These results suggest distinct cell division histories between terminally differentiated neutrophils and macrophages, possibly explaining the early appearance of monocytes in the blood after myeloablative therapy, as compared to neutrophils.

Are fibroblasts involved in joint destruction?

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder that primarily affects the joints and results in the progressive destruction of articular structures, particularly cartilage and bone. Notably, the joint...

Bone cells unite

Bone-resorbing osteoclasts can't pull together in the absence of dendritic cell–specific transmembrane protein (DC-STAMP), according to new data from Yagi and colleagues (page 345). Without this receptor, osteoclast precursor cells cannot fuse with one another. The failure to fuse cripples the bone resorbing function of the cells, causing osteopetrosis (increased bone mass). Figure Fusion of preosteoclasts to generate multinucleated osteoclasts is defective in the absence of DC-STAMP (right). DC-STAMP is a seven-transmembrane–spanning receptor that was originally isolated from dendritic cells (DCs). The ligand for DC-STAMP and its function on DCs are unknown, but this receptor was recently found on osteoclasts and shown to be required for these cells to develop from their macrophage precursors. Yagi and colleagues now refine these data by showing that multinucleated osteoclasts were completely absent in the bones of mice lacking DC-STAMP, although osteoclast development was intact. The DC-STAMP–deficient mice suffered from mild osteopetrosis, as mononucleated osteoclasts can still resorb bone. Whether DC-STAMP triggers fusion directly or triggers the expression of other fusion-inducing receptors remains to be determined. The absence of DC-STAMP also inhibited the formation of foreign body giant cells—products of macrophage fusion that dispose of foreign bodies in tissues. Although the mechanism is not yet known, DC-STAMP may function as a fusion coreceptor in a fashion analogous to the fusion of HIV with target cells, which depends on seven-transmembrane–spanning chemokine receptors (see commentary from Vignery, on page 337).

Macrophages in the murine pancreas and their involvement in fetal endocrine development in vitro

Macrophages are a heterogeneous population of cells that belong to the mononuclear phagocyte system. They play an important role in tissue homeostasis and remodeling and are also potent immune regulators. Pancreatic macrophages are critically involved in the development and pathogenesis of autoimmune diabetes. To elucidate the ontogeny of pancreatic macrophages, we characterized in this study the macrophages present in the adult and developing fetal pancreas of normal mice. We additionally examined the presence of local macrophage precursors and the involvement of macrophages in the growth of endocrine tissue in the fetal pancreas. We identified two phenotypically distinct macrophage subsets in the adult pancreas. The majority of macrophages was CD45(+)ER-MP23(+)MOMA-1(+). Under noninflammatory conditions, only a minority ( approximately 5%) of the pancreatic macrophages additionally expressed the macrophage marker F4/80. In contrast, in the fetal pancreas, phenotypically, mature macrophages were identified exclusively by their expression of F4/80 and lacked detectable staining with ER-MP23 and MOMA-1 antibodies. In fetal pancreas organ cultures, we could show that macrophages develop from pre-existing precursors, which are present in the fetal pancreas at embryonic age 12.5. Moreover, the number of macrophages increased significantly when macrophage-colony stimulating factor was added to these cultures. It is important that this increase of F4/80-positive cells was paralleled by an increase in the number of insulin-producing cells, suggesting that macrophages support the growth of these endocrine cells.

Anatomical Location Determines the Distribution and Function of Dendritic Cells and Other APCs in the Respiratory Tract

APCs, including dendritic cells (DC), are central to Ag surveillance in the respiratory tract (RT). Research in this area is dominated by mouse studies on purportedly representative RT-APC populations derived from whole-lung digests, comprising mainly parenchymal tissue. Our recent rat studies identified major functional differences between DC populations from airway mucosal vs parenchymal tissue, thus seriously questioning the validity of this approach. We addressed this issue for the first time in the mouse by separately characterizing RT-APC populations from these two different RT compartments. CD11c(high) myeloid DC (mDC) and B cells were common to both locations, whereas a short-lived CD11c(neg) mDC was unique to airway mucosa and long-lived CD11c(high) macrophage and rapid-turnover multipotential precursor populations were predominantly confined to the lung parenchyma. Airway mucosal mDC were more endocytic and presented peptide to naive CD4+ T cells more efficiently than their lung counterparts. However, mDC from neither site could present whole protein without further maturation in vitro, or following trafficking to lymph nodes in vivo, indicating a novel mechanism whereby RT-DC function is regulated at the level of protein processing but not peptide loading for naive T cell activation.

Monocyte-derived dendritic cells

It has been recently demonstrated that, in addition to function as macrophage precursors, monocytes have the capacity to differentiate into dendritic cells (DCs), and therefore they play an essential role in both the innate and adaptive immunity. Monocytes display a remarkable functional diversity, allowing them to perform multiple defense functions, from pathogen elimination by phagocytosis, to the induction of antigen-specific T cell responses. This functional potential relies essentially in their developmental plasticity, permitting monocytes to differentiate into different subsets of macrophages and DCs. Although recent data suggest that the acquisition of functional specialization by monocytes is controlled by chemotactic, activation and differentiation factors, how monocyte differentiation occurs under physiological conditions remains largely unknown.

Re-distribution of phospholipase C gamma 2 in macrophage precursors is mediated by the actin cytoskeleton under the control of the Src kinases

Macrophage colony-stimulating factor (M-CSF) is a growth factor that is known to trigger several signalling pathways through receptor tyrosine kinase activation. We investigated the specific requirements for the activation of phospholipase C gamma 2 (PLC-γ2) during the differentiation of mouse bone marrow-derived macrophage precursors. M-CSF stimulation induced rapid PLC-γ2 translocation and phosphorylation from the cytosolic compartment to the cell periphery. Both events were dependent on cytoskeleton integrity and Src kinase activity, but only PLC-γ2 phosphorylation did not require PI3-kinase activity. Biochemical experiments as well as confocal microscopy analyses indicate that the translocation of PLC-γ2 is mediated by the direct association of this protein with the actin cytoskeleton. Using GST-fusion proteins containing various deletions of the PLC-γ2 Src homology region, it was found that PLC-γ2 binds to F-actin via its SH2 domains, a feature that has equally been found in a co-sedimentation assay. This association, which is increased during actin reorganisation and disrupted by cytoskeleton inhibitors, seems to be a primary means to recruit this enzyme to the cell periphery. These results indicate that, upon M-CSF stimulation, PLC-γ2 cellular localisation and phosphorylation are strongly dependent on cytoskeleton architecture of the macrophage precursor as well as the PI3-kinase and the Src kinases.

Altered hematopoiesis in glypican-3-deficient mice results in decreased osteoclast differentiation and a delay in endochondral ossification

Loss of function mutations in the gene encoding the heparan sulfate proteoglycan Glypican-3 ( GPC3) causes an X-linked disorder in humans known as Simpson–Golabi–Behmel Syndrome (SGBS). This disorder includes both pre- and postnatal overgrowth, a predisposition to certain childhood cancers, and a complex assortment of congenital defects including skeletal abnormalities. In this study, we have identified a previously unrecognized delay in endochondral ossification associated with the loss of Gpc3 function. Gpc3 knockout animals show a marked reduction in calcified trabecular bone, and an abnormal persistence of hypertrophic chondrocytes at embryonic day 16.5 (E16.5). These hypertrophic chondrocytes down-regulate Type X collagen mRNA expression and undergo apoptosis, suggesting a normal progression of hypertrophic chondrocyte cell fate. However, replacement of these cells by mineralized bone is delayed in association with a marked delay in the appearance of osteoclasts in the bone in vivo. This delay in vivo correlates with a significant reduction in the capacity to form osteoclasts from bone marrow macrophage precursors in vitro in response to M-CSF and RANKL, and with a reduction in the numbers of bone-marrow-derived cells expressing the markers CD11b and Gr-1. Together, these results indicate selective impairment in the development of the common hematopoietic lineage from which monocyte/macrophages and PMNs are derived. This is the first report of a requirement for heparan sulfate, and specifically Gpc3, in the lineage-specific differentiation of these cell types in vivo.

Chemokines and their Receptors as Targets for the Treatment of COPD

Chronic obstructive pulmonary disease (COPD) is a debilitating respiratory condition, characterised by progressive, irreversible airflow obstruction. The major risk factor for development of COPD is cigarette smoking, and the disease is predicted to become the 3rd leading cause of death by 2020. Currently, there are no pharmacological interventions that halt the progression of COPD; however one strategy is to reduce the chronic lung inflammation associated with this disease. An increased inflammatory infiltrate comprising macrophages, neutrophils and T-lymphocytes is a major hallmark of COPD. Furthermore, both macrophages and neutrophils have the ability to cause all the pathological changes associated with COPD. Chemokines that are elevated in sputum from COPD patients have the capacity to recruit neutrophils, the macrophage precursor cells, monocytes, and T-lymphocytes. Chemokines are considered predominantly chemotactic cytokines however; there is a growing body of evidence demonstrating that chemokines can also act as functional antagonists thus leading to selective recruitment of inflammatory cells. Whilst inhibition of chemokine dependent recruitment of inflammatory cells via small molecule antagonists gives rise to potential treatments for COPD, the discovery that chemokines are also natural antagonists could also be exploited in the ongoing search for treatment of this currently fatal disease. Keywords: copd, chemokines, receptors, therapeutics, cxcr2, cxcr3

Regulation of Human Osteoclast Differentiation by Thioredoxin Binding Protein-2 and Redox-Sensitive Signaling

Differential expression of TBP-2 and Trx-1 occurs during osteoclastogenesis. Adenoviral overexpression of TBP-2 in osteoclast precursors inhibits Trx-1 expression, osteoclast formation, and AP-1 binding activity. TBP-2 and Trx-1 are key regulators of osteoclastogenesis. Thioredoxin binding protein-2 (TBP-2) negatively regulates thioredoxin-1 (Trx-1), a key endogenous modulator of cellular redox and signaling. In gene array analysis, we found that TBP-2 expression was reduced during human osteoclast differentiation compared with macrophage differentiation. Our aim was to determine the roles of TBP-2 and Trx-1 in human osteoclastogenesis and RANKL signaling. Osteoclasts or macrophages were generated from colony-forming unit-granulocyte macrophage (CFU-GM) precursors treated with sRANKL and macrophage-colony-stimulating factor (M-CSF), or M-CSF alone, respectively. Expression of TBP-2 and Trx-1 was quantified by real-time PCR and Western analysis. Adenoviral gene transfer was used to overexpress TBP-2 in precursors. NF-kappaB and activator protein 1 (AP-1) signaling was assessed with EMSA. In the presence of sRANKL, expression of TBP-2 was decreased, whereas Trx-1 expression was increased. The antioxidant N-acetylcysteine reversed this pattern and markedly inhibited osteoclastogenesis. Adenoviral overexpression of human TBP-2 in precursors inhibited osteoclastogenesis and Trx-1 expression, inhibited sRANKL-induced DNA binding of AP-1, but enhanced sRANKL-induced DNA binding of NF-kappaB. These data support significant roles for TBP-2 and the Trx system in osteoclast differentiation that are mediated by redox regulation of AP-1 transcription. A likely mechanism of stress signal induction of bone resorption is provided. Modulators of the Trx system such as antioxidants have potential as antiresorptive therapies.

HIV-1 Nef Promotes Survival of TF-1 Macrophages by Inducing Bcl-XL Expression in an Extracellular Signal-regulated Kinase-dependent Manner

The Nef protein of human immunodeficiency virus-1 (HIV-1) is essential for the progression from human and simian immunodeficiency virus infection to full-blown AIDS. Recent studies indicate that Nef generates anti-apoptotic signals in HIV-infected T cells, suppressing cell death early in infection to allow productive viral replication. Previous work from our laboratory has shown that Nef also promotes proliferation of myeloid cells through a signal transducer and activator of transcription 3-dependent pathway. Here we demonstrate that Nef suppresses cell death induced by cytokine deprivation in the human macrophage precursor cell line, TF-1. Nef selectively induced up-regulation of Bcl-XL, an anti-apoptotic gene that is also regulated by granulocyte/macrophage-colony stimulating factor in this cell line. Activation of the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase pathway also correlated with the survival of TF-1/Nef cells. Using the selective mitogen-activated protein kinase kinase inhibitor PD98059, we found that Nef-induced Erk signaling is essential for Bcl-XL up-regulation and cell survival. In contrast, expression of Bcl-XL and TF-1 survival was not affected by dominant-negative signal transducer and activator of transcription 3. These data suggest that Nef produces survival signals in myeloid cells through Erk-mediated Bcl-XL induction, a pathway distinct from Nef survival pathways recently reported in T lymphocytes.

Oligomerization Is Required for HIV-1 Nef-Induced Activation of the Src Family Protein-Tyrosine Kinase, Hck

Hck is a member of the Src protein-tyrosine kinase family and is expressed strongly in macrophages, an important HIV target cell. Previous studies have shown that Nef, an HIV-1 accessory protein essential for AIDS progression, binds and activates Hck through its SH3 domain. Structural analysis suggests that Nef forms oligomers in vivo, which may bring multiple Hck molecules into close proximity and promote autophosphorylation. Using bimolecular GFP fluorescence complementation, we show for the first time that Nef oligomerizes in living cells and that the oligomers localize to the plasma membrane. To test the role of Nef oligomerization in Hck activation, we fused Nef to the hormone-binding domain of the estrogen receptor (Nef-ER), allowing us to control its dimerization with 4-hydroxytamoxifen (4-HT). In Rat-2 fibroblasts co-expressing Nef-ER and Hck, 4-HT treatment induced Nef-ER dimer and tetramer formation, leading to Hck kinase activation and cellular transformation. The number of transformed foci observed with Nef-ER plus Hck in the presence of 4-HT was markedly greater than that observed with wild-type Nef plus Hck, suggesting that enforced oligomerization enhances activation of Hck by Nef in vivo. Enhanced transformation correlated with increased Hck/Nef complex formation at the plasma membrane. In complementary experiments, we observed that a Nef mutant defective for Hck SH3 domain binding (Nef-PA) suppressed Hck kinase activation and transformation by the wild-type Hck/Nef complex. This effect correlated with the formation of a ternary complex between wild-type Nef, Nef-PA, and Hck, suggesting that Nef-PA suppresses Hck activation by blocking wild-type Nef oligomerization. Finally, Nef-ER induced Hck activation in a 4-HT-dependent manner in the macrophage precursor cell line TF-1, suggesting that oligomerization is essential for signaling through Hck in a cell background relevant to HIV infection. Together, these data demonstrate that Nef oligomerization is critical to the activation of Hck in vivo, and suggest that inhibitors of oligomerization may suppress Nef signaling through Hck in HIV-infected macrophages, slowing disease progression.

Electron microscopy of tissue adherent to explanted electrodes in dystonia and Parkinson's disease.

Deep brain stimulation (DBS) is used to treat a variety of severe medically intractable movement disorders, including Parkinson's disease, tremor and dystonia. There have been few studies examining the effect of chronic DBS on the brains of Parkinson's disease patients. Most of these post mortem studies concluded that chronic DBS caused mild gliosis around the lead track and did not damage brain tissue. There have been no similar histopathological studies on brains from dystonic patients who have undergone DBS. In this study, our objective was to discover whether tissue would be attached to DBS electrodes removed from patients for routine clinical reasons. We hoped that by examining explanted DBS electrodes using scanning (SEM) and/or transmission (TEM) electron microscopy we might visualize any attached tissue and thus understand the electrode-human brain tissue interaction more accurately. Initially, SEM was performed on one control DBS electrode that had not been implanted. Then 21 (one subthalamic nucleus and 20 globus pallidus internus) explanted DBS electrodes were prepared, after fixation in 3% glutaraldehyde, for SEM (n = 9) or TEM (n = 10), or both (n = 2), according to departmental protocol. The electrodes were sourced from two patients with Parkinson's disease, one with myoclonic dystonia, two with cervical dystonia and five with primary generalized dystonia, and had been in situ for 11 and 31 months (Parkinson's disease), 16 months (myoclonic dystonia), 14 and 24 months (cervical dystonia) and 3-24 months (primary generalized dystonia). Our results showed that a foreign body multinucleate giant cell-type reaction was present in all TEM samples and in SEM samples, prewashed to remove surface blood and fibrin, regardless of the diagnosis. Some of the giant cells were >100 microm in diameter and might have originated from either fusion of parenchymal microglia, resident perivascular macrophage precursors and/or monocytes/macrophages invading from the blood stream. The presence of mononuclear macrophages containing lysosomes and sometimes having conspicuous filopodia was detected by TEM. Both types of cell contained highly electron-dense inclusions, which probably represent phagocytosed material. Similar material, the exact nature of which is unknown, was also seen in the vicinity of these cells. This reaction was present irrespective of the duration of implantation and may be a response to the polyurethane component of the electrodes' surface coat. These findings may be relevant to our understanding of the time course of the clinical response to DBS in Parkinson's disease and various forms of dystonia, as well as contributing to the design characteristics of future DBS electrodes.

The in and out of monocytes in atherosclerotic plaques: Balancing inflammation through migration

Recent clinical and experimental evidence indicates that inflammatory processes in the vascular wall are the decisive factor that accounts for the rate of lesion formation and clinical development in patients suffering from atherosclerosis (1). The response-to-injury hypothesis (2) predicts that particular stimuli (= injury) trigger the initiation of an inflammatory cascade in the vessel wall. In addition, alteration of the lipid metabolism is a critical factor intertwined with the complex inflammatory reactions that occur during lesion development. The progressive accumulation of macrophages and other immune cells in the atherosclerotic plaque is one of the hallmarks in atherogenesis (Fig. 1). The ins and outs of macrophage precursors, the blood monocyte, in atherosclerotic lesions have been elegantly dissected in a study by Llodra et al. (3) in this issue of PNAS. The authors suggest that not only recruitment of monocytes to but also their retention within the atherosclerotic lesion contributes to the progression of plaque development.

Towards a transcriptome definition of microglial cells

This study provides an expression signature of interferon-gamma (IFN-gamma)-activated microglia. Microglia are macrophage precursor cells residing in the brain and spinal cord. The microglial phenotype is highly plastic and changes in response to numerous pathological stimuli. IFN-gamma has been established as a strong immunological activator of microglial cells both in vitro and in vivo. Affymetrix RG_U34A microarrays were used to determine the effect of IFN-gamma stimulation on migroglia cells isolated from newborn Lewis rat brains. More than 8,000 gene sequences were examined, i.e., 7,000 known genes and 1,000 expressed sequence tag (EST) clusters. Under baseline conditions, microglia expressed 326 of 8,000 genes examined (approximately 4% of all genes, 182 known and 144 ESTs). Transcription of only 34 of 7,000 known genes and 8 of 1,000 ESTs was induced by IFN-gamma stimulation. The majority of the newly expressed genes encode pro-inflammatory cytokines and components of the MHC-mediated antigen presentation pathway. The expression of 60 of 182 identified genes and of 9 of 144 ESTs was increased by IFN-gamma, whereas 29 of 182 known genes and 7 of 144 ESTs were down-regulated or undetectable in IFN-gamma-stimulated cultures. Overall, the activating effect of IFN-gamma on the microglial transcriptome showed restriction to pathways involved in antigen presentation, protein degradation, actin binding, cell adhesion, apoptosis, and cell signaling. In comparison, down-regulatory effects of IFN-gamma stimulation appeared to be confined to pathways of growth regulation, remodeling of the extracellular matrix, lipid metabolism, and lysosomal processing. In addition, transcriptomic profiling revealed previously unknown microglial genes that were de novo expressed, such as calponin 3, or indicated differential regulatory responses, such as down-regulation of cathepsins that are up-regulated in response to other microglia stimulators.

Diversity in Phenotype and Steroid Hormone Dependence in Dendritic Cells and Macrophages in the Mouse Uterus1

The dendritic cells and related antigen-presenting cells (APCs) that activate lymphocytes for acquired immunity in the female reproductive tract are not well characterized. The aim of the present study was to examine heterogeneity among uterine APCs in mice and, specifically, to determine whether phenotypically and functionally distinct subpopulations of dendritic cells and macrophages can be identified. Using immunohistochemistry, abundant cells expressing APC-restricted molecules major histocompatibility complex (MHC) class II, F4/80, class A scavenger receptor, macrosialin, and sialoadhesin were evident in estrous mice. Cells expressing the costimulatory molecule B7-2 were rarely observed. Flow cytometric analysis revealed three subpopulations of uterine APCs. Undifferentiated macrophages were F4/80-positive (+), MHC class II-negative (-) cells, of which 70-80% expressed CD11b, but few expressed class A scavenger receptor, macrosialin, or sialoadhesin. Mature macrophages were F4/80+/MHC class II+ cells, of which approximately 50% expressed CD11b, class A scavenger receptor, macrosialin, and sialoadhesin. Uterine dendritic cells were F4/ 80-/MHC class II+ cells, with stimulatory immunoaccessory function relative to uterine macrophages and heterogeneous expression of dendritic markers 33D1, DEC205, CD11c, and CD1. Experiments in ovariectomized mice showed that undifferentiated macrophages were steroid hormone dependent but that mature macrophages and dendritic cells persisted after depletion of ovarian steroid hormones, although with altered phenotypes. In summary, our findings identify three discrete populations of APCs inhabiting the murine uterus and suggest that both mature macrophages and dendritic cells differentiate from undifferentiated macrophage precursor cells. Plasticity in the ontogenetic and functional relationships between uterine dendritic cells and macrophages likely is critical in regulating immune responses conducive to reproductive success.