Resident Peritoneal Research Articles

MHCII+ Macrophages in Peritoneal and Pleural Cavities Require IRF4 for Development from Circulating Monocytes

Abstract Resident macrophages in peritoenal and pleural cavities exist as two distinct populations: F4/80+ICAM2+macrophages and MHCII+ macrophages. F4/80+ ICAM2+macrophages are derived from CX3CR1+embryonic precursors, and maitained by the transcription factor Gata6. However, the origin and regulating factors that maintain MHCII+ macrophages remain unknown. Here, we show that the MHCII+ macrophages arise postnatally from monocytes, which continuously replenish this subset through adulthood. Gene expression analysis identified distinct surface markers such as CD226 and revealed that the transcription factor IRF4 was selectively expressed in these macrophages relative to other organs. Monocytes first enter peritoneal or pleural cavities to become MHC II+ cells that up-regulate CD226 later as they continue to mature. In the absence of IRF4 or after administration of oral antibiotics, MHCII+CD226−CD11c− monocyte-derived cells accumulated in peritoneal and pleural cavities, but CD11c+ CD226+ macrophages were lost. Thus, MHC II+ resident peritoneal and pleural macrophages are continuously replenished by blood monocytes recruited to the peritoneal and pleural cavities constitutively, starting after birth, where they require IRF4 and signals likely derived from the microbiome to fully differentiate.

Trabectedin Re-Educates Resting Peritoneal Macrophages into M1 Subtype

Macrophages play an important role in the inflammatory response, acting as antigen-presenting cells and modifying the cytokine milieu and the intensity of T-cell signaling. The electrophysiological properties of macrophages depend on their state of functional activation. Trabectedin induces rapid apoptosis exclusively in mononuclear phagocytes and this effect contributes directly to its antitumor activity. The aim of this study was to evaluate the effects of trabectedin on KV and Kir channels in resident peritoneal and bone marrow-derived macrophages (BMDM) under both short- and long-term incubation with the drug. Experiments were performed using the whole-cell configuration of the patch-clamp technique. Following exposure (ca. 10 min) of peritoneal macrophages or BMDM to 100 nM trabectedin neither KV nor Kir were modified. The electrophysiological effects of prolonged treatment with trabectedin were studied after 16 h of incubation with the drug. Under these conditions, trabectedin produced a concentration-dependent effect that was qualitatively similar to the response observed after LPS challenge. Moreover, the increase in KV observed appeared in parallel with a use-dependent decline, typical of macrophages stimulated with LPS. Similarly, the degree of inactivation of the current was greater and faster at higher trabectedin concentrations. All these results suggest that: 1) Trabectedin does not interact with Kir or KV channel. 2) Trabectedin acts by re-educating mouse resting peritoneal macrophages into M1-subtype. Supported by PharmaMar, SAF2013-45800-R and FIS RD12/0042/0019 Grants.

MHC II+ resident peritoneal and pleural macrophages rely on IRF4 for development from circulating monocytes.

Peritoneal and pleural resident macrophages in the mouse share common features and in each compartment exist as two distinct subpopulations: F4/80(+) macrophages and MHC II(+) CD11c(+) macrophages. F4/80(+) macrophages derive from embryonic precursors, and their maintenance is controlled by Gata6. However, the origin and regulatory factors that maintain MHC II(+) macrophages remain unknown. Here, we show that the MHC II(+) macrophages arise postnatally from CCR2-dependent precursors that resemble monocytes. Monocytes continuously replenish this subset through adulthood. Gene expression analysis identified distinct surface markers like CD226 and revealed that the transcription factor IRF4 was selectively expressed in these macrophages relative to other organs. Monocytes first entered peritoneal or pleural cavities to become MHC II(+) cells that up-regulated CD226 and CD11c later as they continued to mature. In the absence of IRF4 or after administration of oral antibiotics, MHC II(+)CD226(-)CD11c(-) monocyte-derived cells accumulated in peritoneal and pleural cavities, but CD11c(+) CD226(+) macrophages were lost. Thus, MHC II(+) resident peritoneal and pleural macrophages are continuously replenished by blood monocytes recruited to the peritoneal and pleural cavities constitutively, starting after birth, where they require IRF4 and signals likely derived from the microbiome to fully differentiate.

Interferon-gamma and nitric oxide synthase 2 mediate the aggregation of resident adherent peritoneal exudate cells: implications for the host response to pathogens.

Interferon-gamma (Ifnγ), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/nitric oxide adduct (DETA/NO), and Nos2 -/- mice identified Nitric oxide (NO) induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with Nos2 -/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or “group behavior” of APECs are discussed in the context of host resistance to infectious organisms.

Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids

Macrophage phagocytosis of apoptotic cells, or unopsonized viable CD47−/− red blood cells, can be mediated by the interaction between calreticulin (CRT) on the target cell and LDL receptor-related protein-1 (LRP1/CD91/α2-macroglobulin receptor) on the macrophage. Glucocorticoids (GC) are powerful in treatment of a range of inflammatory conditions, and were shown to enhance macrophage uptake of apoptotic cells. Here we investigated if the ability of GC to promote macrophage uptake of apoptotic cells could in part be mediated by an upregulation of macrophage LRP1 expression. Using both resident peritoneal and bone marrow-derived macrophages, we found that the GC dexamethasone could dose- and time-dependently increase macrophage LRP1 expression. The GC receptor–inhibitor RU486 could dose-dependently prevent LRP1 upregulation. Dexamethasone-treated macrophages did also show enhanced phagocytosis of apoptotic thymocytes as well as unopsonized viable CD47−/− red blood cells, which was sensitive to inhibition by the LRP1-agonist RAP. In conclusion, these data suggest that GC-stimulated macrophage uptake of apoptotic cells may involve an upregulation of macrophage LRP1 expression and enhanced LRP1-mediated phagocytosis.

TLR Agonists That Induce IFN-β Abrogate Resident Macrophage Suppression of T Cells

Resident tissue macrophages (Mφs) continually survey the microenvironment, ingesting Ags and presenting them on their surface for recognition by T cells. Because these Ags can be either host cell- or pathogen-derived, Mφs must be able to distinguish whether a particular Ag should provoke an immune response or be tolerated. However, the mechanisms that determine whether Mφs promote or inhibit T cell activation are not well understood. To investigate this, we first determined the mechanism by which murine resident peritoneal Mφs suppress in vitro T cell proliferation in the absence of pathogens and then explored the effects of different pathogen-derived molecules on Mφ immunosuppression. Our results suggest that, in response to IFN-γ, which is secreted by TCR-activated T cells, resident peritoneal Mφs acquire immunosuppressive properties that are mediated by NO. However, pretreatment of Mφs with LPS or dsRNA, but not CpG or peptidoglycan, eliminates their suppressive properties, in part via the induction of autocrine-acting IFN-β. These results suggest TLR agonists that activate TRIF, and consequently induce IFN-β, but not those that exclusively signal through MyD88, abrogate the immunosuppressive properties of Mφs, and thus promote T cell expansion and elimination of invading microorganisms.

Resident Peritoneal Inflammatory Cells are Pivotal in the Development of Experimental Atherosclerosis

Based on evidence that ionizing radiation can ameliorate chronic and autoimmune diseases in patients and experimental animals, we investigated the effects of radiation on the induction and development of experimental atherogenesis. Male New Zealand rabbits were divided into 5 groups and given an atherogenic diet for 90 days. Peritoneal and thoracic areas (9 Gy) were irradiated on the 1st and 45th days for groups 1 and 2, the 45th day for groups 3 and 4, and not at all for group 5. Prior to irradiation, the peritoneal cavity of animals from groups 1 and 3 was washed with buffered saline. Cells collected by peritoneal washing were reinfused into the peritoneal cavity of the same animal after irradiation. Animals from groups 2 and 4 were intraperitoneally injected with saline as a control. Despite similar lipid profiles among the experimental groups, the percentage of aortas covered by plaques was remarkably reduced (p<0.001) among animals submitted to irradiation (groups 2 and 4). These differences were completely abolished in irradiated animals reconstituted with their own peritoneal cells. These findings point to an important role of resident inflammatory peritoneal cells in experimental atherogenesis.

NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A

Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.

Cytoplasmic Linker Protein-170 Enhances Spreading and Phagocytosis in Activated Macrophages by Stabilizing Microtubules

Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-gamma and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated alpha-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-gamma/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-gamma/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.

Characterization and modeling of monocyte‐derived macrophages after spinal cord injury

Spinal cord injury (SCI) elicits a neuroinflammatory reaction dominated by microglia and monocyte-derived macrophages (MDM). Because MDM do not infiltrate the spinal cord until days after injury, it may be possible to control whether they differentiate into neuroprotective or neurotoxic effector cells. However, doing so will require better understanding of the factors controlling MDM differentiation and activation. Our goal was to develop an in vitro model of MDM that is relevant in the context of SCI. This tool would allow future studies to define mechanisms and intracellular signaling pathways that are associated with MDM-mediated neuroprotection or neurotoxicity. We first characterized SCI-induced cytokine expression in MDM using laser capture microdissection and real-time PCR. Based on this data, we assessed which easily procurable primary macrophage subset would mimic this phenotype in vitro. We established the baseline and inductive potential of resident peritoneal, thioglycollate-elicited peritoneal and bone marrow-derived macrophages (BMDM) at the molecular, cellular and functional level. Of these cells, only BMDM retained the phenotypic, molecular and functional characteristics of MDM that infiltrate the injured spinal cord. Thus, peripheral macrophages should not be used interchangeably in vitro to model the functional consequences of the MDM response elicited by SCI.

Trem-Like Transcript 2 Is Expressed on Cells of the Myeloid/Granuloid and B Lymphoid Lineage and Is Up-Regulated in Response to Inflammation

The triggering receptor expressed on myeloid cells (TREM) gene cluster encodes a group of transmembrane proteins that are emerging as important components in innate and adaptive immunity. In both mice and humans, the TREM gene cluster encodes eight receptors; only four of these, however, are direct homologs: TREM-1, TREM-2, TREM-like transcript 1 (TLT1), and TLT2. Of the transmembrane receptors encoded by the four conserved genes within this cluster, TLT2 has not been studied previously. Data presented in this study demonstrate that TLT2 is expressed early in B cell development in conjunction with B220 and is detected on all developing mouse B cell populations as well as B cells in the periphery. TLT2 expression on B cells in the periphery exhibits a distinct hierarchy with the highest detectable levels observed on B1 B cells in the peritoneum. The overall gradation of TLT2 expression on B cells is: B1 > marginal zone/transitional 2 > transitional 1 > follicular. Additionally, TLT2 expression was observed on mouse neutrophils throughout the body. Although monocytes were not observed to express TLT2, resident peritoneal and lung macrophages do express TLT2, suggesting that it is up-regulated in association with terminal differentiation of monocytes. Finally, both neutrophils and macrophages were observed to up-regulate TLT2 expression in vivo in response to inflammatory stimuli, whereas TLT2 expression on B cells remained unchanged. In conclusion, the data suggest that TLT2 may be involved in the innate immune response based on its expression profile and the fact that it is up-regulated in response to inflammation.

IgE expression on the surface of B1 and B2 lymphocytes in experimental murine schistosomiasis

In a previous study we monitored the distribution and phenotype expression of B1 cells during the evolution of experimental murine schistosomiasis mansoni and we proposed that the B1 cells were heterogeneous: a fraction which originated in the spleen and followed the migratory pathway to mesenteric ganglia, while the other was the resident peritoneal B1-cell pool. In the present study, we have addressed the question of whether these two B1-lymphocyte populations are involved in the production of the late Ig isotype IgE, which is present in high levels in schistosomal infection. Lymphocyte expression of surface markers and immunoglobulins were monitored by immunofluorescence flow cytometry. Both in the spleen and mesenteric ganglia, the B1 and B2 cells were induced to switch from IgM to IgE in the early Th2-dominated phase of the disease, with an increase of IgE in its later phases. Conversely, peritoneal B1-IgM+ switched to the remaining IgE+ present in high numbers in the peritoneal cavity throughout the disease. We correlated the efficient induction of the expression of late Ig isotypes by B1 cells with high levels of inflammatory cytokines due to the intense host response to the presence of worms and their eggs in the abdominal cavity. In conclusion, B1 cells have a different switch behavior from IgM to IgE indicating that these cell sub-populations depend on the microenvironment.

Recognition and phagocytosis of apoptotic T cells by resident murine tissue macrophages require multiple signal transduction events

Macrophages (Mø) ingest apoptotic cells with unique effects on their cytokine production, but the signaling pathways involved are virtually unknown. Signal transduction in response to recognition of apoptotic thymocytes by resident murine alveolar (AMø) or peritoneal (PMø) Mø was studied by in vitro phagocytosis assay. Phagocytosis was decreased in a dose-dependent and nontoxic manner by inhibiting phosphatidylinositol 3 kinase (wortmannin and LY294002), protein tyrosine phosphorylation (herbimycin A, genistein, piceatannol, and for AMø only, PP2), and protein kinase C (staurosporine, Gö 6976, and calphostin C). Exposure of Mø to apoptotic or heat-killed thymocytes, but not to viable thymocytes, activated ERK1/2 rapidly, as detected by specific phosphorylation, but did not activate NF-kappaB or MAP kinases p38 or JNK. Mø phagocytosis of apoptotic T cells requires tyrosine, serine/threonine, and lipid phosphorylation. Mø recognition of apoptotic T cells triggers rapid but limited MAP kinase activation.

Procoagulant and anticoagulant activities of resident and inflammatory peritoneal cells.

In the present study, the involvement of both procoagulants and anticoagulants from adherent peritoneal cells in an inflammatory model was investigated. Mice were injected with thioglycollate broth or lipopolysaccharide (LPS). Cells were harvested by peritoneal lavage. Adherent peritoneal cells were cultured further +/- LPS. Subsequently, their ability to activate either Factor X or prothrombin, and their ability to inactivate thrombin through a thrombin-degrading mast cell chymase, was assayed. Inflammatory cells expressed reduced amounts of thrombin-inactivating activity as compared with control resident peritoneal cells. Both resident and inflammatory cells expressed potent prothrombinase activities. Further stimulation of the various cellular populations with LPS in vitro had very little effect on the prothrombinase and thrombin-inactivating activities, bud had a strong stimulatory effect on the Factor X-activating activities.

Exposure of macrophages to an enzymatically inactive macrophage mannose receptor ligand augments killing of Candida albicans.

Macrophages (Mphi) are involved in host defenses against opportunistic pathogens. Previous studies by the present investigators indicate that Mphi exposed to enzymatically active myeloperoxidase (MPO), exhibited both increased phagocytosis and killing of Candida albicans. The purpose of this study was to determine if enzymatically inactive Mphi-mannose receptor (MMR) ligands could function similarly. Resident murine peritoneal Mphi were exposed to the MMR ligands, mannosylated bovine serum albumin (mBSA), and enzymatically inactive myeloperoxidase (iMPO), followed by exposure to opsonized C. albicans. Both mBSA and iMPO induced a slight increase in the number of phagocytizing cells; however, candidacidal activity was significantly higher in treated cultures compared to controls (P < or = 0.001). The production of reactive oxygen intermediates (ROI) was detected using chemiluminescence. After employment of ROI scavengers, a decrease in candidacidal activity was observed. The data suggest that MMR-ligand interaction alone is sufficient to significantly enhance the candidacidal activity of Mphi via ROI, and that iMPO which is released at a site of inflammation induces Mphi-mediated killing of microorganisms. These findings indicate a previously unrecognized role of iMPO.

Differential regulation of macrophage arginine metabolism: a proposed role in wound healing

Nitric oxide (NO) and ornithine, products of NO synthase or arginase, respectively, have opposing biological activities. The effect of mediators of leukocyte activation and inhibition on arginine metabolism of resident mouse peritoneal exudate cells (MPEC) was determined. Factors that increased basal NO synthase activity, interferon (IFN)-gamma and lipopolysaccharide (LPS), decreased arginase activity in intact cells. Transforming growth factor (TGF)-beta1 decreased IFN-gamma-stimulated NO synthase activity and produced a reciprocal increase in urea and ornithine release. TGF-beta1 had no effect on the activity of these enzymes in LPS-stimulated MPEC. Corticosterone (Cort, 100 ng/ml) decreased the basal activity of both enzymes. However, Cort inhibited NO synthase activity and increased ornithine release in MPEC exposed to IFN-gamma or LPS. The difference between arginase activity in intact cells vs. that of cell lysates suggested intracellular inhibition of arginase activity. Products of NO synthase, NO and citrulline, were shown to inhibit MPEC arginase activity under maximal assay conditions. Intracellular pH was not altered by exposure of MPEC to LPS, IFN-gamma, TGF-beta, and Cort. This reciprocal change in arginine metabolism is proposed to be an important component of wound healing. Expression of NO synthase creates a cytotoxic environment that may be important to the early phase of wound healing. As wound healing progresses, increased arginase activity produces an environment favorable for fibroblast replication and collagen production.

The capsular polysaccharide complex of Bacteroides fragilis induces cytokine production from human and murine phagocytic cells

To stimulate early-phase immunologic events following Bacteroides fragilis infection in the peritoneal cavity, we examined the cytokine response of several cell types to purified capsular polysaccharide complex (CPC) and lipopolysaccharide (LPS) of this organism. Cytokines were produced from murine resident peritoneal (MRP) cells as well as human peripheral blood leukocytes. MRP cells cocultured with either B. fragilis CPC of LPS in vitro produced tumor necrosis factor alpha and interleukin-1alpha (IL-1alpha). In addition, MRP cells challenged with CPC produced IL-10. Human peripheral blood monocytes and polymorphonuclear leukocytes secreted IL-8 when cultured in the presence of CPC.

Regulation of Lysophospholipase Activity of the 85-kDa Phospholipase A2 and Activation in Mouse Peritoneal Macrophages

The regulation of the lysophospholipase activity of the 85-kDa cytosolic phospholipase A2 (PLA2) was studied in vitro and in stimulated macrophages. Bovine serum albumin was found to inhibit lysophospholipase activity of the recombinant 85-kDa PLA2 when assayed at a relatively low substrate concentration. Inhibition could be reversed if the substrate concentration was increased or if Ca2+ was present in the assay. Incubation of recombinant enzyme with macrophage membranes and lipid extracts from macrophage membranes resulted in the release of arachidonic acid, as well as, stearic acid, which is enriched at the sn-1 position of macrophage phospholipids. This suggests that with a bilayer substrate the PLA2 can sequentially deacylate the sn-2 then sn-1 acyl groups. This was verified by demonstrating that the phospholipids, phosphatidylcholine and phosphatidylinositol, were hydrolyzed to glycerophosphocholine and glycerophosphoinositol by incubation with recombinant 85-kDa PLA2. The 85-kDa enzyme was identified as the main lysophospholipase activity in mouse peritoneal macrophage cytosols. Addition of Ca2+ to the assay enhanced activity, but this effect decreased as the substrate concentration was increased. Incubation of macrophages with zymosan increased the lysophospholipase activity of the 85-kDa PLA2 in cytosols. Phosphorylation of recombinant PLA2 with mitogen-activated protein kinase resulted in an increase in lysophospholipase, as well as, PLA2 activity. In macrophages stimulated with zymosan release of stearic acid (18:0) and palmitic acid (16:0) was observed in addition to arachidonic acid (20:4). These results are consistent with a role of the 85-kDa PLA2 in regulating lysophospholipid levels in macrophages during zymosan stimulation.

The potential role of the macrophage colony-stimulating factor, CSF-1, in inflammatory responses: characterization of macrophage cytokine gene expression.

In this report we report that recombinant human monocyte-macrophage colony-stimulating factor-1 (CSF-1) induces resident murine peritoneal cells (PCs) to transcribe several inflammatory cytokine genes, including interleukin (IL)-1 alpha, IL-1 beta, IL-6, and granulocyte-macrophage CSF in a dose-dependent and time-related manner. Peak mRNA levels were seen between 4 and 6 h. CSF-1 did not modulate the expression of tumor necrosis factor-alpha mRNA. The serum content of the culture medium appeared to regulate both the extent of CSF-1-induced gene transcription and the adherence properties of the cells. Decreasing the serum concentration significantly reduced CSF-1-induced transcription and was associated with the rapid spreading of the majority of the adherent cells. This reduced sensitivity to CSF-1 was paralleled by a markedly lower levels of c-fms mRNA encoding the CSF-1 receptor. Induced gene transcription was followed by the release of large quantities of IL-6 only. IL-1 activity remained associated with the cells. Neither supernatant nor cell lysate granulocyte-macrophage CSF activity was inducible above the low levels associated with control cultures. Evidence that the mononuclear phagocytes, as opposed to B or T cells, were the targets of CSF-1 was obtained in two ways: (1) PCs from B6 scid/scid and NOD scid/scid mice consisting of 78-86% MAC-1+, F4/80+ cells and few B or T cells, as shown by flow cytometry analysis, released 5- to 10-fold more IL-6 in response to CSF-1 stimulation than B6 PCs, which contained approximately 30% double-positive cells, and (2) pretreatment of B6 PCs with antibodies to the CSF-1 receptor blocked the CSF-1-induced secretion of IL-6. These data suggest that CSF-1 primes noninflammatory mononuclear phagocytes for a role in inflammatory responses but does not provide the necessary signals for either secretion or translation of all cytokines equally.

The monoclonal antibody ER-BMDM1 recognizes a macrophage and dendritic cell differentiation antigen with aminopeptidase activity.

Here we describe the reactivity of monoclonal antibody (mAb) ER-BMDM1, directed against a 160-kDa cell membrane-associated antigen (Ag) with amino-peptidase activity. The aminopeptidase recognized by ER-BMDM1 is present on various mouse macrophage (M phi) and dendritic cell (DC) subpopulations as well as on microvillous epithelia. Analysis of ER-BMDM1 Ag expression in in vitro models of M phi maturation revealed that the Ag is expressed at increasing levels upon maturation of M phi. In vivo, high level expression of the ER-BMDM1 Ag occurs after the monocytic stage of maturation, since bone marrow cells and peripheral blood monocytes are essentially ER-BMDM1 negative. Analysis of isolated-resident and elicited M phi populations showed that ER-BMDM1 recognizes a specific subpopulation of mature M phi: only some resident peritoneal and alveolar M phi are ER-BMDM1 positive, whereas virtually all thioglycollate-elicited peritoneal exudate M phi bind the mAb. In lymphoid organs, a subpopulation of M phi is recognized as well as interdigitating cells (IDC) located in T cell areas. Phenotypic analysis of isolated DC--the in vitro equivalents of IDC--from spleen and lymph nodes confirmed that the majority of this important antigen-presenting cell population expresses the ER-BMDM1 aminopeptidase. The molecular characteristics of the ER-BMDM1 Ag suggest that it may represent the mouse homolog of human CD13.