Ingestion Of Apoptotic Cells Research Articles

Leukocyte Transglutaminase 2 Expression Limits Atherosclerotic Lesion Size

Transglutaminase 2 (TG2), a broadly expressed regulator of protein cross-linking, wound healing, and tissue fibrosis, mediates apoptotic cell ingestion and transforming growth factor-beta release by macrophages and thereby can limit leukocyte-mediated inflammation. In atherosclerosis, oxidative stress and accumulation of unesterified cholesterol stimulate atherosclerotic lesion cell apoptosis. Cell death in advanced atherosclerotic lesions promotes lesion expansion and vulnerable plaques prone to rupture. Hence, we tested the hypothesis that leukocyte TG2 expression limits atherosclerosis. We transplanted TG2-/- or TG2+/+ bone marrow into lethally irradiated low-density lipoprotein receptor (LDLR)-/- mice and evaluated diet-induced atherosclerosis after 16 weeks. We subsequently studied cultured TG2-/- and congenic TG2+/+ mouse macrophages for selected atherogenesis regulatory functions. Atherosclerotic aortic valve lesions in LDLR-/- recipients of TG2-/- bone marrow were larger and more subintimal lesional macrophage penetration than in TG2+/+ marrow recipients. Lesion intimal TG2 expression appeared robust in TG2+/+ but not TG2-/- marrow recipients. Cultured TG2-/- macrophages demonstrated diminished phagocytosis of apoptotic leukocytes, unaltered endocytosis, and degradation of oxidized LDL but decreased retinoic acid induction of the reverse cholesterol transport and apoptotic cell uptake mediator ABCA1. We conclude that macrophage TG2 expression promotes both apoptotic cell clearance and ABCA1 expression in vitro and limits atherosclerotic lesion size in vivo.

Roles of Ras and extracellular signal-regulated kinase-dependent IκBα degradation in oridonin-enhanced phagocytosis of apoptotic cells by human macrophage-like U937 cells

Rapid recognition and ingestion of apoptotic cells by phagocytes are important for the prevention of toxic intracellular contents release, thereby attenuate inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). We have reported that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic U937 cells by macrophage-like U937 cells through TNFα and IL-1β release. In this study, the molecular mechanisms involved in this phagocytic process are investigated. Inhibitors of Ras and Raf1 kinase significantly reduced oridonin-induced phagocytic stimulation as well as extracellular signal-regulated kinase (ERK) phosphorylation. Simultaneously, oridonin-enhanced engulfment was partially blocked by a nuclear factor (NF)-κB inhibitor PDTC or proteasome inhibitor MG132. Further studies revealed that oridonin induced IκBα degradation, which was prevented by Ras inhibitor manumycin A, ERK inhibitor PD98059, but not prevented by c-Jun N-terminal kinase (JNK) MAPK inhibitor SP600125, and up-regulated expression of IL-1β precursor. These results demonstrate that Ras/Raf1/ERK signaling pathway-dependent IκBα degradation, resulting in NF-κB activation, participates in regulation of oridonin-enhanced phagocytosis, and one of its effector functions is to induce synthesis of IL-1β, which partially contribute to phagocytic activity of oridonin.

Clearance of apoptotic cells is not impaired in mouse embryos deficient in class A scavenger receptor types I and II (CD204)

Elimination of apoptotic cells is an important mechanism to maintain proper embryonal morphogenesis. The class A scavenger receptor type I, II (CD204), one of the major receptors expressed on macrophages, is a receptor actively involved in recognition and ingestion of apoptotic cells. To clarify the role of CD204 in embryonic morphogenesis, we performed immunohistochemical and immunoelectron microscopic studies using CD204-deficient mouse embryos. In control mice, almost all macrophages expressed CD204 from embryonic day 9.5 (E9.5). Phagocytes engulfing dead cells in the E13.5 interdigit region showed strong expression of CD204, indicating that CD204 was actively involved in apoptotic cell clearance. However, CD204 is not essential for the embryonic clearance of apoptotic cells, because CD204-deficient embryos developed normally without any retardation in footplate remodeling. Up-regulation of CD36 in CD204-deficient fetal macrophages suggested that CD36 substitutes for CD204 function. We also found that mesenchymal cells frequently engulfed apoptotic cells especially in early embryonal stages. These data suggest that CD204 is partially but not essentially involved in apoptotic cell clearance in embryogenesis. During early embryonal development, mesenchymal cells, rather than macrophages, play a major role in apoptotic cell clearance.

Monocytes Recruited to the Lungs of Mice during Immune Inflammation Ingest Apoptotic Cells Poorly

Apoptotic cells must be cleared to resolve inflammation, but few resident alveolar macrophages (AMo) from normal lungs ingest apoptotic cells. We examined how Mo ingestion of apoptotic cells is altered during immune inflammation induced by intratracheal challenge of primed C57BL/6 mice using sheep red blood cells. Resident AMo were labeled in situ before challenge using intravenous PKH26 to distinguish them from recruited monocytes. Using flow cytometry, we identified phagocytosis of fluorescently-labeled apoptotic thymocytes by alveolar mononuclear phagocytes in vitro and in vivo, and measured surface molecule expression. Intratracheal challenge induced rapid recruitment of monocytes, peaking at Day 3 and decreasing thereafter, whereas numbers of resident AMo did not change significantly. At all times, the percentage of phagocytes ingesting apoptotic thymocytes in vitro was greater among resident AMo (28-45%) than among recruited monocytes (9-19%), but was low in both cell types relative to ingestion of immunoglobulin-opsonized targets. There was also a nonsignificant trend toward lower ingestion by monocytes in vivo. MerTK, a receptor tyrosine kinase crucial for apoptotic cell phagocytosis, was expressed by resident AMo, but not by recruited monocytes. Relative to resident AMo, monocytes recruited to the alveolus ingest apoptotic cells meagerly, possibly due to absence of MerTK expression.

Chronically stimulated microglial cells do no longer alter their immune functions in response to the phagocytosis of apoptotic cells

In an autoimmune inflammatory setting, ingestion of apoptotic T cells leads to a down-regulation of microglial immune functions. Recent studies have indicated that microglia can be matured by exposure to GM-CSF. GM-CSF stimulation led to a differentiated microglial phenotype and enhanced antigen-presenting capabilities. The secretion of TNF-α was significantly decreased by the uptake of apoptotic cells in unstimulated microglia, but not in GM-CSF-differentiated microglia. IL-10 secretion was unaffected. After ingestion of apoptotic cells, only previously unstimulated, but not GM-CSF-differentiated microglial cells decreased their T cell-activating potential as measured by IFN-γ secretion in antigen-activated MBP-specific T cells. Thus, GM-CSF stimulation reduces the immunomodulatory functions of microglial cells.

Distinct maturation of, but not migration between, human monocyte-derived dendritic cells upon ingestion of apoptotic cells of early or late phases.

Cell death via apoptosis is a normal physiological process. Rapid, but silent, removal of apoptotic cells (ACs) plays an essential role in maintaining homeostasis in the immune system. Defective clearance of ACs allows ACs to accumulate and undergo late phase apoptosis, also known as secondary necrosis, which may generate danger signals, leading to inflammation or autoimmunity. In this study we investigate the outcome of dendritic cells (DCs), which are potent APCs, on the interaction with ACs of early or late phase. Immature DCs internalized ACs of both early and late phases with similar efficiency. However, DCs that had taken up ACs of early phase acquired a non-fully mature DC phenotype, expressing low MHC class II complex, costimulatory molecule CD40, and mature DC-restricted marker CD83, and had a low capacity to stimulate allogeneic CD4+ T cell proliferation, whereas DCs that had taken up ACs of late phase acquired a mature DC phenotype with enhanced T cell stimulatory capacity. Ingestion of either early or late ACs induced minimal production of IL-12 and modulated CC chemokine and CCR expression in DCs. In particular, there was down-regulation of CCR5 and up-regulation of CCR7, resulting in switches in responsiveness from inflammatory to lymphoid chemokines. We conclude from these data that after taking up ACs of either early or late phases, DCs acquire the capability of homing to draining lymph nodes, and the distinct maturation between DCs taking up early or late ACs may contribute to DC function in the induction of T cell tolerance or Ag-specific T cell response, respectively.

Resident murine alveolar and peritoneal macrophages differ in adhesion of apoptotic thymocytes.

Apoptotic cells must be cleared efficiently by macrophages (Mø) to prevent autoimmunity, yet their ingestion impairs Mø microbicidal function. The principal murine resident lung phagocyte, the alveolar Mø (AMø), is specifically deficient at apoptotic cell ingestion, both in vitro and in vivo, compared with resident peritoneal Mø (PMø). To further characterize this deficiency, we assayed static adhesion in vitro using apoptotic thymocytes and resident AMø and PMø from normal C57BL/6 mice. Adhesion of apoptotic thymocytes by both types of Mø was rapid, specific, and cold-sensitive. Antibody against the receptor tyrosine kinase MerTK (Tyro12) blocked phagocytosis but not adhesion in both types of Mø. Surfactant protein A increased adhesion and phagocytosis by AMø, but not to the levels seen using PMø. Adhesion was largely cation-independent for PMø and calcium-dependent for AMø. Adhesion was not inhibited in either Mø type by mAbs against beta1 or beta3 integrins or scavenger receptor I/II (CD204), but AMø adhesion was inhibited by specific mAbs against CD11c/CD18. Thus, resident murine tissue Mø from different tissues depend on qualitatively disparate receptor systems to bind apoptotic cells. The decreased capacity of murine AMø to ingest apoptotic cells is only partially explained by reduced initial adhesion.

An SH2 Domain-dependent, Phosphotyrosine-independent Interaction between Vav1 and the Mer Receptor Tyrosine Kinase: A MECHANISM FOR LOCALIZING GUANINE NUCLEOTIDE-EXCHANGE FACTOR ACTION

Mer belongs to the Mer/Axl/Tyro3 receptor tyrosine kinase family, which regulates immune homeostasis in part by triggering monocyte ingestion of apoptotic cells. Mutations in Mer can also cause retinitis pigmentosa, again due to defective phagocytosis of apoptotic material. Although, some functional aspects of Mer have been deciphered, how receptor activation lead to the physiological consequences is not understood. By using yeast two-hybrid assays, we identified the carboxyl-terminal region of the guanine nucleotide-exchange factor (GEF) Vav1 as a Mer-binding partner. Unlike similar (related) receptors, Mer interacted with Vav1 constitutively and independently of phosphotyrosine, yet the site of binding localized to the Vav1 SH2 domain. Mer activation resulted in tyrosine phosphorylation of Vav1 and release from Mer, whereas Vav1 was neither phosphorylated nor released from kinase-dead Mer. Mutation of the Vav1 SH2 domain phosphotyrosine coordinating Arg-696 did not alter Mer/Vav1 constitutive binding or Vav1 tyrosine phosphorylation but did retard Vav1 release from autophosphorylated Mer. Ligand-dependent activation of Mer in human monocytes led to Vav1 release and stimulated GDP replacement by GTP on RhoA family members. This unusual constitutive, SH2 domain-dependent, but phosphotyrosine-independent, interaction and its regulated local release and subsequent activation of Rac1, Cdc42, and RhoA may explain how Mer coordinates precise cytoskeletal changes governing the ingestion of apoptotic material by macrophages and pigmented retinal epithelial cells.

Syk activation is a leukotriene B4–regulated event involved in macrophage phagocytosis of IgG-coated targets but not apoptotic cells

AbstractMacrophages are called upon to ingest both IgG-coated targets and apoptotic cells. Important roles for tyrosine kinase Syk and leukotriene B4 (LTB4) are recognized in FcγR-mediated phagocytosis. Here we evaluated the roles of Syk and LTB4 in macrophage phagocytosis of apoptotic thymocytes versus IgG-coated erythrocytes. Macrophage ingestion of apoptotic thymocytes was not influenced by exogenous or endogenous LTB4 nor associated with Syk activation (phosphorylation). By contrast, LTB4 dose-dependently amplified FcγR-mediated phagocytosis as well as Syk activation. Furthermore, a role for endogenous LTB4 in Syk activation during FcγR-mediated phagocytosis was demonstrated using pharmacologic and genetic abrogation of 5-lipoxygenase. LTB4 was unique among 5-lipoxygenase products in this regard, since LTD4 and 5-hydroxyeicosatetraenoic acid (HETE) were unable to amplify Syk activation in response to FcγR engagement. Ca2+ chelation studies revealed that FcγR-mediated Syk activation as well as LTB4 amplification thereof was Ca2+ regulated. These 2 parallel phagocytic processes therefore exhibit initial divergence in signal transduction events, with Syk activation being an LTB4-regulated event in FcγR-mediated but not apoptotic cell ingestion. As LTB4 is an important proinflammatory product of macrophages, we speculate that this divergence evolved to permit FcγR-mediated phagocytosis to proceed in an inflammatory milieu, while apoptotic cell clearance is noninflammatory.

The mer receptor tyrosine kinase: expression and function suggest a role in innate immunity.

The mer receptor tyrosine kinase mediates phagocytosis of apoptotic cells and modulates cytokine production; it is also required for prevention of systemic autoimmune disease. Using a mer-specific antibody, we have confirmed the presence of mer on macrophages and now report its expression on NK cells, NKT cells, and dendritic cells (DC). We found that DC do not require mer for ingestion of apoptotic cells, as DC from mer-deficient mice phagocytose apoptotic cells normally. Mer was observed in splenic sections on cells outside follicular areas, probably representing DC and macrophages. Mer apparently participates in NKT-cell antigen-induced signaling, as NKT cells from mer-deficient mice evinced much lower cytokine production after in vivo alpha-galactosylceramide stimulation; this defect was intrinsic to the mer-deficient NKT cells. Taken together, these studies show mer expression on cells of the innate immune system. Mer, through its binding of lipid antigens, may not only mediate ingestion of apoptotic cells, but also signal events in NK cells, NKT cells, and DC.

Histidine-rich glycoprotein binds to DNA and Fc gamma RI and potentiates the ingestion of apoptotic cells by macrophages.

Histidine-rich glycoprotein (HRG) is an abundant serum protein that exhibits many functions in diverse biological systems. In this study, we show that HRG potentiates the ingestion of apoptotic cells by mature human monocyte-derived macrophages (HMDM). HRG bound specifically to apoptotic Jurkat cells and mature HMDM in a saturable and concentration-dependent manner. Purified HRG or HRG in sera increased the number of HMDM-containing apoptotic cells and accelerated the ingestion, while neutralization or depletion of HRG from sera reduced this effect. Anti-FcgammaRI mAb inhibited HRG binding to HMDM, while DNA, but not chromatin, inhibited HRG binding to apoptotic cells, and either anti-FcgammaRI or DNA abrogated the HRG-dependent ingestion. The findings indicate that HRG, by acting as a bridge between DNA on apoptotic cells and FcgammaRI on HMDM, is a key physiological mediator of apoptotic cell clearance by macrophages.

Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex.

Removal of cells dying by apoptosis is essential to normal development, maintenance of tissue homeostasis, and resolution of inflammation. Surfactant protein A (SP-A) and surfactant protein D (SP-D) are high abundance pulmonary collectins recently implicated in apoptotic cell clearance in vitro. Other collectins, such as mannose-binding lectin and the collectin-like C1q, have been shown to bind to apoptotic cells and drive ingestion through interaction with calreticulin and CD91 on the phagocyte in vitro. However, only C1q has been shown to enhance apoptotic cell uptake in vivo. We sought to determine the relative importance of SP-A, SP-D, and C1q in pulmonary clearance of apoptotic cells using knockout and overexpressing mice, and to determine the role of calreticulin and CD91 in this process. SP-A, SP-D, and C1q all enhanced apoptotic cell ingestion by resident murine and human alveolar macrophages in vitro. However, only SP-D altered apoptotic cell clearance from the naive murine lung, suggesting that SP-D plays a particularly important role in vivo. Similar to C1q and mannose-binding lectin, SP-A and SP-D bound to apoptotic cells in a localized, patchy pattern and drove apoptotic cell ingestion by phagocytes through a mechanism dependent on calreticulin and CD91. These results suggest that the entire collectin family of innate immune proteins (including C1q) works through a common receptor complex to enhance removal of apoptotic cells, and that collectins are integral, organ-specific components of the clearance machinery.

Expression of phosphatidylserine receptor and down-regulation of pro-inflammatory molecule production by its natural ligand in rat microglial cultures.

Exposure of phosphatidylserine (PS), an aminophospholipid normally sequestered in the inner leaflet of plasma membrane, is one of the crucial steps in the recognition and ingestion of apoptotic cells by macrophages. The recognition of PS on apoptotic cells by peripheral macrophages is mediated by a phosphatidylserine-specific receptor (PtdSerR), which has recently been cloned. In spite of the important role of apoptosis in the CNS, the process of apoptotic neuron recognition by microglia is poorly understood. Because recent studies suggest that engagement of PS with a not yet characterized microglial receptor is necessary for apoptotic neuron uptake, we investigated the expression of PtdSer-R and its functional role in neonatal rat brain microglial cultures. Semi-quantitative RT-PCR analysis revealed that PtdSerR mRNA was detectable in unstimulated cultures and enhanced in LPS activated microglia. The presence of PS-liposomes strongly reduced the release of pro-inflammatory molecules such as nitric oxide, interleukin-1beta, and tumor necrosis factor-alpha by LPS-activated microglia. At variance, the immunoregulatory cytokines interleukin-10 and transforming growth factor-beta1 were moderately decreased or unaffected. The activity of PS-liposomes was mimicked by the PS head group phospho-L-serine, but not by phosphatidylcholine-containing liposomes. Our data suggest that, as for peripheral macrophages, PS through its receptor can modulate microglial activation toward an anti-inflammatory phenotype.

Inhibitory effects of apoptotic cell ingestion upon endotoxin-driven myeloid dendritic cell maturation.

Dendritic cells (DCs) are the sentinels of the immune system, able to interact with both naive and memory T cells. The recent observation that DCs can ingest cells dying by apoptosis has raised the possibility that DCs may, in fact, present self-derived Ags, initiating both autoimmunity and tumor-specific responses, especially if associated with appropriate danger signals. Although the process of ingestion of apoptotic cells has not been shown to induce DC maturation, the exact fate of these phagocytosing DCs remains unclear. In this paper we demonstrate that DCs that ingest apoptotic cells are able to produce TNF-alpha but have a diminished ability to produce IL-12 in response to external stimuli, a property that corresponds to a failure to up-regulate CD86. By single-cell analysis we demonstrate that these inhibitory effects are restricted to those DCs that have engulfed apoptotic cells, with bystander DCs remaining unaffected. These changes were independent of the production of anti-inflammatory cytokines TGF-beta1 and IL-10 and corresponded with a diminished capacity to stimulate naive T cells. Thus, the ingestion of apoptotic cells is not an immunologically null event but is capable of modulating DC maturation. These results have important implications for our understanding of the role of clearance of dying cells by DCs not only in the normal resolution of inflammation but also in control of subsequent immune responses to apoptotic cell-derived Ags.

Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation.

Ingestion of apoptotic cells in vitro by macrophages induces TGF-beta1 secretion, resulting in an anti-inflammatory effect and suppression of proinflammatory mediators. Here, we show in vivo that direct instillation of apoptotic cells enhanced the resolution of acute inflammation. This enhancement appeared to require phosphatidylserine (PS) on the apoptotic cells and local induction of TGF-beta1. Working with thioglycollate-stimulated peritonea or LPS-stimulated lungs, we examined the effect of apoptotic cell uptake on TGF-beta1 induction. Viable or opsonized apoptotic human Jurkat T cells, or apoptotic PLB-985 cells, human monomyelocytes that do not express PS during apoptosis, failed to induce TGF-beta1. PS liposomes, or PS directly transferred onto the PLB-985 surface membranes, restored the TGF-beta1 induction. Apoptotic cell instillation into LPS-stimulated lungs reduced proinflammatory chemokine levels in the bronchoalveolar lavage fluid (BALF). Additionally, total inflammatory cell counts in the BALF were markedly reduced 1-5 days after apoptotic cell instillation, an effect that could be reversed by opsonization or coinstillation of TGF-beta1 neutralizing antibody. This reduction resulted from early decrease in neutrophils and later decreases in lymphocytes and macrophages. In conclusion, apoptotic cell recognition and clearance, via exposure of PS and ligation of its receptor, induce TGF-beta1 secretion, resulting in accelerated resolution of inflammation.

Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation

Ingestion of apoptotic cells in vitro by macrophages induces TGF-β1 secretion, resulting in an anti-inflammatory effect and suppression of proinflammatory mediators. Here, we show in vivo that direct instillation of apoptotic cells enhanced the resolution of acute inflammation. This enhancement appeared to require phosphatidylserine (PS) on the apoptotic cells and local induction of TGF-β1. Working with thioglycollate-stimulated peritonea or LPS-stimulated lungs, we examined the effect of apoptotic cell uptake on TGF-β1 induction. Viable or opsonized apoptotic human Jurkat T cells, or apoptotic PLB-985 cells, human monomyelocytes that do not express PS during apoptosis, failed to induce TGF-β1. PS liposomes, or PS directly transferred onto the PLB-985 surface membranes, restored the TGF-β1 induction. Apoptotic cell instillation into LPS-stimulated lungs reduced proinflammatory chemokine levels in the bronchoalveolar lavage fluid (BALF). Additionally, total inflammatory cell counts in the BALF were markedly reduced 1–5 days after apoptotic cell instillation, an effect that could be reversed by opsonization or coinstillation of TGF-β1 neutralizing antibody. This reduction resulted from early decrease in neutrophils and later decreases in lymphocytes and macrophages. In conclusion, apoptotic cell recognition and clearance, via exposure of PS and ligation of its receptor, induce TGF-β1 secretion, resulting in accelerated resolution of inflammation.

Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation

Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation

Microglial phagocytosis of apoptotic inflammatory T cells leads to down-regulation of microglial immune activation.

Apoptotic cell death is an established mechanism to terminate an inflammatory response in rodent or human brains. Microglia, as the resident phagocyte, is a strong candidate for the clearance of apoptotic lymphocytes. Apoptosis was induced in cultured autologous thymocytes and in myelin basic protein (MBP)-specific, encephalitogenic T cells from Lewis rats by the addition of 0.1 microg/ml methylprednisolone. The amount of phagocytosis of apoptotic cells was assessed using an in vitro phagocytosis assay. Supernatants were collected to measure microglial cytokine secretion. The state of immune activation in microglia was investigated by a T cell proliferation assay and by flow cytometric analysis of microglial surface expression of immune molecules. Microglia ingested specifically apoptotic cells (apoptotic thymocytes as well as MBP-specific T cells) in contrast to nonapoptotic control cells (p < 0.0001). Subsequent secretion of the proinflammatory cytokines TNF-alpha and IL-12 was significantly decreased, while the secretion of IL-10 and TGF-beta was not affected. Furthermore, ingestion of apoptotic cells led to increased microglial MHC class II expression without concomitant increase in MHC class I, costimulatory molecules, and ICAM expression. The Ag-specific activation of MBP-specific T cells in cocultures with microglia that had ingested apoptotic cells was significantly less than that of identical T cells that interacted with nonphagocytosing microglia. Together with negative results obtained in a trans-well system, this is in support of a cell contact-mediated effect. Microglia might play an important role in the clearance of apoptotic cells. The uptake of apoptotic cells by microglia is tolerogenic and results in a reduced proinflammatory cytokine production and a reduced activation of encephalitogenic T cells. This might help to restrict an autoimmune inflammation and minimize damage in the inflamed brain.

Transforming Growth Factor-β1 Secretion From Murine Macrophages Is Released by In Vivo Ingestion of Apoptotic Cells

(CHEST 2001; 120:3S)

Transcriptional and Translational Regulation of Inflammatory Mediator Production by Endogenous TGF-β in Macrophages That Have Ingested Apoptotic Cells

Abstract We recently reported that phagocytosis of apoptotic cells inhibits the release of inflammatory cytokines by human macrophages. In this paper we show that apoptotic cell uptake by mouse J774 macrophages also inhibits the synthesis and secretion of the chemokines, macrophage inflammatory protein-2 (Mip-2), KC, and Mip-1α (but not that of monocyte chemoattractant protein-1 (MCP-1)/JE), and increases TGF-β formation. Anti-TGF-β neutralizing Abs largely reversed the inhibitory effect of apoptotic cell uptake, and accordingly, exogenous TGF-β down-regulated the synthesis of the same mediators. Apoptotic cell ingestion or TGF-β also inhibited Mip-2 and Mip-1α gene expression in LPS-treated J774 cells, whereas TNF-α mRNA levels were unaffected. Importantly, TGF-β pretreatment of J774 cells did not significantly alter chemokine and TNF mRNA stability. Finally, we found that apoptotic cell uptake and TGF-β did not modulate NF-κB or AP-1 DNA binding in J774 cells. We conclude that the decreased production of chemokines and TNF resulting from apoptotic cell ingestion is largely mediated by a common event, i.e., feedback inhibition by endogenous TGF-β, but involves different mechanisms. Whereas TNF-α production appears to be translationally down-regulated, the suppression of most chemokines investigated appears to reflect transcriptional inhibition. In a broader context, the impairment of chemokine and TNF generation by apoptotic cell uptake might represent an important mechanism contributing to the resolution of inflammation. An additional consequence could be the selective recruitment of monocytes into inflammatory sites, as MCP-1/JE production by mouse macrophages was unaffected by apoptotic cell uptake, in contrast to other chemokines.