Effects of macrophage colony-stimulating factor (M-CSF) on protease production from monocyte, macrophage and foam cell in vitro: a possible mechanism for anti-atherosclerotic effect of M-CSF

M-CSF is a growth factor that stimulates proliferation and differentiation of monocyte/macrophage-lineage cells. In our previous studies, M-CSF regresses atherosclerotic lesions preformed in aorta of high cholesterol-fed rabbit. Immunohistochemical analysis indicated that extracellular matrix (ECM), such as collagen, was especially eliminated in the intima of atherosclerotic lesion. To define the collagen-lowering potential of M-CSF, we have studied the effects of M-CSF on production of collagen-degrading proteases, such as MMP-1, -9 and urokinase in vitro. Monocytes freshly isolated from human peripheral blood produced MMP-9, but not urokinase, and M-CSF enhanced MMP-9 production. Macrophages were prepared by culturing monocytes for 10 days in the presence or absence of M-CSF, and protease production was assayed. M-CSF augmented production of MMP-9 and urokinase in a dose-dependent manner. M-CSF also enhanced MMP-1 production of macrophages, but not significantly. Foam cells were prepared by culturing macrophages in the presence of acetyl LDL, and protease production from these cells were also elevated by M-CSF. These results suggest that M-CSF exogenously administered in atherosclerotic rabbits might regress the thickened intima by activating macrophages to degrade collagen accumulated in the lesion.     

Characterization of monocyte/macrophage subsets in the skin and peripheral blood derived from patients with systemic sclerosis

Introduction

Recent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.

Methods

Skin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.

Results

In the skin from SSc patients, the number of CD163⁺ cells or CD204⁺ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14⁺ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14⁺ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163⁺ cells belong to CD14^brightCD204⁺ population.

Conclusions

This is the first report indicating CD163⁺ or CD204⁺ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14^brightCD163⁺CD204⁺ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163⁺ or CD204⁺ macrophages in the skin.     

Coordinate expression of OPN and associated receptors during monocyte/macrophage differentiation of HL-60 cells

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits α_v, β₃, β₁, were examined. Results were as follows: (1) PMA induced OPN mRNA and OPN secretion into media; (2) untreated cells expressed β₁ and CD44 mRNA, and PMA induced α_v and β₃ mRNA and increased β₁ and CD44 mRNA expression; (3) PMA increased levels of α_v, β₃, β₁ and CD44 protein on the cell surface; and (4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, α_v, β₃, β₁, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells. J. Cell. Physiol. 175:229–237, 1998. © 1998 Wiley-Liss, Inc.     

Gomesin acts in the immune system and promotes myeloid differentiation and monocyte/macrophage activation in mouse

Due to the cytotoxic effect of antimicrobial peptides (AMP) against several microorganism and tumor cells has been proposed their association with the immune system. However, just a few reports have shown this relationship. In this study, mice were treated with gomesin, a β-hairpin AMP that exhibit high cytotoxicity against bacterial and tumor cells. Different effects in the immune system were observed, such as, decrease of CD3⁺ in T lymphocytes (Control: 17.7 ± 1.4%; Gomesin: 7.67 ± 1.2%) and in hematopoietic progenitors and increase of hematopoietic stem cell (Control: 0.046 ± 0.004%; Gomesin: 0.067 ± 0.003%), B220⁺ B lymphocytes (Control: 38.63 ± 1.5%; Gomesin: 47.83 ± 0.48%), and Mac-1⁺F4/80⁺ macrophages (Control: 11.76 ± 3.4%; Gomesin: 27.13 ± 4.0%). Additionally, macrophage increase was accompanied by an increase of macrophage phagocytosis (Control 20.85 ± 1.53; Gomesin 31.32 ± 1 Geometric mean), interleukin 6 (Control: 47.24 ± 1.9 ng/mL; Gomesin: 138.68 ± 33.68 ng/mL) and monocyte chemoattractant protein-1 (Control: 0.872 ± 0.093 ng/mL; Gomesin: 1.83 ± 0.067 ng/mL). Thus, this report showed immunomodulatory activity of gomesin in the immune system of mice.     

Induced expression and association of the Mona/Gads adapter and Gab3 scaffolding protein during monocyte/macrophage differentiation

Mona/Gads is a Grb2-related, Src homology 3 (SH3) and SH2 domain-containing adapter protein whose expression is restricted to cells of hematopoietic lineage (i.e., monocytes and T lymphocytes). During monocyte/macrophage differentiation, Mona is induced and interacts with the macrophage colony-stimulating factor receptor, M-CSFR (also called Fms), suggesting that Mona could be involved in developmental signaling downstream of the M-CSFR by recruiting additional signaling proteins to the activated receptor. Our present results identify Mona as a specific partner protein for the DOS/Gab family member Gab3 in monocytic/macrophage development. Mona does not interact with Gab2; however, Gab3 also forms a complex with the Mona-related adapter Grb2. Glutathione S-transferase pull-down experiments demonstrate that the Mona and Gab3 interaction utilizes the carboxy-terminal SH3 domain of Mona and the atypical proline-rich domain of Gab3. Mona is known to interact with the phosphorylated Y697 site of the M-CSFR. The M-CSFR mutation Y697F exhibited qualitative and quantitative abnormalities in receptor and Gab3 tyrosine phosphorylation, and Mona induction was greatly reduced. The Y807F M-CSFR mutation is defective in differentiation signaling, but not growth signaling, and also fails to induce Mona protein expression. During M-CSF-stimulated macrophage differentiation of mouse bone marrow cells, Mona and Gab3 expression is coinduced, these proteins interact, and Mona engages in multimolecular complexes. These data suggest that association of Mona and Gab3 plays a specific role in mediating the M-CSFR differentiation signal.     

Carbohydrate and peptide antigens in macrophage populations derived from human bone marrow and milk: an immunomorphological and immunochemical analysis

Summary An immunomorphological and immunochemical study was performed to elucidate the pattern of carbohydrate antigens and their relationships to the cluster differentiation (CD) 68 epitopes on macrophages derived from human bone marrow and milk. Core and backbone antigens recognized by lectins fromBauhinia purpurea (BPA),Helix pomatia (HPA),Arachis hypogaea (PNA),Glycine max. (SBA),Griffonia simplicifolia (GSA-I-B₄),Lycopersicon esculentum (LEA) andErythrina cristagalli (ECA) were expressed by both macrophage populations. Additionally, they exhibited various peripheral type 1 and type 2 carbohydrate antigens. In bone marrow trephine biopsies, the number of macrophages stained by the CD68-specific monoclonal antibody PG-M1 exceeded significantly (range 30–40%) the subpopulation expressing SBA, GSA-I-B₄ and ECA binding sites as well as the Lewis^a antigen. This result is very interesting since, fromin vitro studies, GSA-I-B₄ and SBA are known to react especially with activated macrophages. Western blotting experiments on milk macrophage lysates revealed that ECA, GSA-I-B₄, BPA, PNA and MAA visualize a 110 kDa band isographic with the CD68 antigen detected by PG-M1, KP1 and Ki-M1P monoclonal antibodies. These antibodies recognize peptide epitopes as shown by enzyme-linked immunosorbent assays after biochemical modification of milk macrophage lysates. This result is in keeping with the assumption that the CD68 antigen consists of a highly glycosylated mucin-type glycoprotein comprising various differentiation-dependent epitopes.     

Lipid droplet: A functionally active organelle in monocyte to macrophage differentiation and its inflammatory properties

Lipid droplets (LDs) perform several important functions like inflammatory responses, membrane trafficking, acts as secondary messengers, etc. rather than simply working as an energy reservoir. LDs have been implicated as a controlling factor in the progression of atherosclerosis followed by foam cell formation that derives from macrophages during the differentiation process. However, the role of LDs in monocyte differentiation or its further immunological function is still an area that mandates in-depth investigation. We report that LD dynamics is important for differentiation of monocytes and is absolutely required for sustained and prolonged functional activity of differentiated macrophages. In THP-1 cell line model system, we elucidated that increase in total LD content in monocyte by external lipid supplements, can induce monocyte differentiation independent of classical stimuli, PMA. Differential expression of PLIN2 and ATGL during the event, together with abrogation of de novo lipogenesis further confirmed the fact. Besides, an increase in LD content by free fatty acid supplement was able to exert a synergistic effect with PMA on differentiation and phagocytic activity compared to when they are used alone. Additionally, we have shown Rab5a to play a vital role in LDs biosynthesis/maturation in monocytes and thereby directly affecting differentiation of monocytes into macrophages via AKT pathway. Thus our study reveals the multi-faceted function of LDs during the process of monocyte to macrophage differentiation and thereby helping to maintain the functional activity.     

Accessory cell function during monocyte/macrophage differentiation: relation to interleukin-1 (IL-1 beta) production and release

Human peripheral blood monocytes (Mo) can differentiate into highly active accessory cells and approach the phenotype and function of dendritic cells instead of developing into macrophages (Mph). Here we report that monocyte-derived accessory cells (m-AC), but not Mph, spontaneously synthesize and release high amounts of interleukin-1 (IL-1 beta). Furthermore, m-AC retained a high T-cell stimulatory activity and a non-macrophagic phenotype for at least 12 days in culture. They were shown to be weakly adherent, non-phagocytic, and most of them were negative for nonspecific esterase. In contrast, Mo differentiating into mature Mph only transiently showed an elevated accessory function but at no time appeared to release intracellular IL-1 beta into the supernatant when cultured in the absence of exogenous triggers. Additionally, they gained a high phagocytic capacity and a strong expression of Fc-receptors within 4 days. Addition of lipopolysaccharides (LPS) to Mph stimulated IL-1 beta release but concomitantly led to a strong reduction of the Mph-phenotype. Thus, the release of IL-1 beta from monocyte-derived cells negatively correlated with the expression of the Mph phenotype but did not necessarily correlate with their accessory function. These observations may reflect an antagonistic regulation of Mph phenotype and cytokine release in cells of the monocytic lineage and suggest that IL-1 beta release is not essential for accessory activity but might serve rather as an autocrine signal to prolong the accessory function of m-AC.     

Coexistence resulting from an alternation of density dependent and density independent growth

When two species compete with each other, one is likely to displace or exclude the other. Several circumstances under which they may coexist indefinitely have been presented in the literature; the present contribution presents examples of one more. Under circumstances where both populations are repeatedly decreased (for example because of annual environmental changes) then subsequent to each decrease both species grow unrestrictedly and then interact with each other in a competitive fashion. If the species that grows more rapidly under unrestricted conditions is at a disadvantage during competitive phases of growth, this effect prolongs coexistence but may not prevent eventual extinction of one or the other species. However, it is shown that there are certain broad ranges of conditions for population growth that lead to permanent cyclical stability. The stability described here is such that the ecosystem will return to the same dynamic balance even when severely perturbed. It is also shown that this kind of stability can be either favored or prevented in certain cases by random fluctuations in the environment affecting season length, kill factor, etc.     

Thymocyte differentiation activity from the cloned monocyte/macrophage cell line RAW 264.7. Alterations in the expression of immature thymocyte surface antigens

The cloned monocyte/macrophage cell line RAW 264.7 was previously shown to produce thymocyte mitogenic and co-mitogenic activity that eluted from a Sephadex G-75 column not only at approximately 16,000 daltons, the m.w. described for interleukin 1 (IL 1), but also at 30,000 to 40,000 daltons. The studies reported here indicate that the 30,000 to 40,000 dalton molecule has thymic differentiating activity. Thymocytes from A/J mice were fractionated on discontinuous BSA gradients, which yielded populations of cells enriched for immature and mature cells. The cells found at the interface between 35 and 29% BSA (band 1 cells), which are the most immature, were cultured for 48 hr with highly purified IL 1, with the 30,000 to 40,000 dalton form of thymocyte co-mitogenic activity obtained after Sephadex G-75 chromatography and chromatofocusing chromatography, or with media alone. The surface antigens TL-3, H-2Kk, Thy-1.2, Lyt-1, and Lyt-2 were examined by immunofluorescence. It was found that the highly purified 30,000 to 40,000 dalton species of co-mitogenic activity induced a significant increase in the content of surface H-2Kk, a decrease in TL-3, and a very small decrease in Thy-1.2 on the cell surface, whereas IL 1 was not capable of inducing a change in these surface antigens. There was no change in Lyt-1 on the surface of band 1 thymocytes after incubation with either IL 1 or the 30,000 to 40,000 dalton species. The 30,000 to 40,000 dalton species caused a significant decrease in the percentage of cells staining positive for Lyt-2, whereas IL 1 caused a smaller but significant decrease in Lyt-2. These changes in the surface markers TL-3, H-2Kk, and Thy-1.2 are consistent with changes that occur during thymocyte differentiation. It was also observed that the proliferative response to the 30,000 to 40,000 dalton form and IL 1 increased with increasing functional maturity of each band of thymocytes when used in the thymocyte mitogenic assay. However, only the 30,000 to 40,000 dalton form was capable of inducing a proliferative response in the immature band 1 thymocytes in the thymocyte co-mitogenic assay. These results indicate that the RAW 264.7 cells produce a factor that has, in addition to thymocyte co-mitogenic activity, thymocyte differentiation activity, and this factor is distinct from IL 1.     

Prefrontal cortex and somatosensory cortex in tactile crossmodal association: an independent component analysis of ERP recordings

Our previous studies on scalp-recorded event-related potentials (ERPs) showed that somatosensory N140 evoked by a tactile vibration in working memory tasks was enhanced when human subjects expected a coming visual stimulus that had been paired with the tactile stimulus. The results suggested that such enhancement represented the cortical activities involved in tactile-visual crossmodal association. In the present study, we further hypothesized that the enhancement represented the neural activities in somatosensory and frontal cortices in the crossmodal association. By applying independent component analysis (ICA) to the ERP data, we found independent components (ICs) located in the medial prefrontal cortex (around the anterior cingulate cortex, ACC) and the primary somatosensory cortex (SI). The activity represented by the IC in SI cortex showed enhancement in expectation of the visual stimulus. Such differential activity thus suggested the participation of SI cortex in the task-related crossmodal association. Further, the coherence analysis and the Granger causality spectral analysis of the ICs showed that SI cortex appeared to cooperate with ACC in attention and perception of the tactile stimulus in crossmodal association. The results of our study support with new evidence an important idea in cortical neurophysiology: higher cognitive operations develop from the modality-specific sensory cortices (in the present study, SI cortex) that are involved in sensation and perception of various stimuli.     

Alterations in cellular proteome and secretome upon differentiation from monocyte to macrophage by treatment with phorbol myristate acetate: Insights into biological processes

Monocyte and macrophage are mainly involved in immune response and inflammatory processes. Monocytes circulate in the bloodstream and migrate to various tissues where they can differentiate to macrophages. However, the molecular basis of biological processes involved in this cellular differentiation remains ambiguous. This study was to investigate alterations in cellular and secreted proteins after this differentiation phase. Macrophage was differentiated from U937 human monocytic cell line by treatment with 100 ng/ml phorbol myristate acetate (PMA) for 48 h. Cellular and secreted proteins extracted from PMA-treated cells (macrophages) were compared with those of untreated cells (monocytes) using 2-DE (n = 5 gels/condition; stained with Deep Purple fluorescence dye). Quantitative intensity analysis revealed 81 and 67 protein spots whose levels were significantly altered in cellular proteome and secretome. These proteins were subsequently identified by Q-TOF MS and/or MS/MS analyses. The altered levels of cellular elongation factor-2 (EF-2) and secreted α-tubulin were confirmed by Western blot analysis. Global protein network analysis demonstrated that these altered proteins were involved in cell death, lipid metabolism, cell morphology, cellular movement, and protein folding. Our data may provide some insights into molecular mechanisms of biological processes upon differentiation from monocytes to macrophages.     

Quantitative analysis of depolarization-induced ATP release from mouse brain synaptosomes: External calcium dependent and independent processes

Summary We and others have shown previously that ATP is secreted from mouse brain synaptosomes following depolarization of the membrane by high [K⁺] ₀ and the time course can be monitored accurately by measuring the light emitted from luciferin-luciferase included in the reaction medium. In the present work we have evaluated the relative importance of [Ca²⁺] ₀ and membrane potential on the ATP secretion process by modelling the time course of ATP release under different conditions. After correction of the records for destruction of released ATP by synaptosomal ecto-ATPase activity, we found that ATP secretion occurs by an apparent first order process. We also established that, in addition to the classical [Ca²⁺] ₀ -dependent mode, ATP secretion also occurred in the absence of extracellular calcium ([Ca²⁺] ₀ < 1 m). Upon lowering the extracellular Ca²⁺ concentration, both the rate and the extent of ATP secretion decreased. To assess the contribution of membrane potential to the release rate we measured ATP secretion at membrane potentials determined by extracellular [K⁺] ₀ (or [Rb⁺] ₀ ) as defined by the distribution of the carbocyanine dye, diSC₃(5). Rate constants computed from measured secretion curves revealed that this parameter was essentially independent of membrane potential in the absence of [Ca²⁺] ₀ . Noise analysis of the light signal showed that the variance increased upon stimulation by high [K⁺] ₀ , suggesting that both modes of secretion are quantal. Thus, we conclude that the rate of ATP secretion from nerve terminals depends upon Ca²⁺ entry but not on membrane potential, per se     

Saliva from Lutzomyia longipalpis induces CC chemokine ligand 2/monocyte chemoattractant protein-1 expression and macrophage recruitment

Saliva of bloodfeeding arthropods has been incriminated in facilitating the establishment of parasite in their host. We report on the leukocyte chemoattractive effect of salivary gland homogenate (SGH) from Lutzomyia longipalpis on saliva-induced inflammation in an air pouch model. SGH (0.5 pair/animal) was inoculated in the air pouch formed in the back of BALB/c or C57BL/6 mice. L. longipalpis SGH induced a significant influx of macrophages in BALB/c but not in C57BL/6 mice. SGH-induced cell recruitment reached a peak at 12 h after inoculation and was higher than that induced by the LPS control. This differential cell recruitment in BALB/c mice was directly correlated to an increase in CCL2/MCP-1 expression in the air pouch lining tissue. In fact, treatment with bindarit, an inhibitor of CCL2/MCP-1 synthesis, and also with a specific anti-MCP-1 mAb resulted in drastic reduction of macrophage recruitment and inhibition of CCL2/MCP-1 expression in the lining tissue. CCL2/MCP-1 production was also seen in vitro when J774 murine macrophages were exposed to L. longipalpis SGH. The SGH effect was abrogated by preincubation with serum containing anti-SGH IgG Abs as well as in mice previously sensitized with L. longipalpis bites. Interestingly, the combination of SGH with Leishmania chagasi induced an increased recruitment of neutrophils and macrophages when compared with L. chagasi alone. Taken together these results suggest that SGH not only induces the recruitment of a greater number of macrophages by enhancing CCL2/MCP-1 production but also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation.     

Single-cell sequencing analysis and transcriptome analysis constructed the macrophage related gene-related signature in lung adenocarcinoma and verified by an independent cohort

BackgroundRecent studies have emphasized the close relationship between macrophages and tumor immunity, and the prognosis of lung adenocarcinoma (LUAD) patients is intimately linked to this. Nonetheless, the prognostic signature and classification of different immune patterns in LUAD patients based on the macrophages is largely unexplored.MethodsTwo sc-RNAseq datasets of LUAD patients were collected and reprocessed. The differentially expressed genes (DEGs) related to macrophages between LUAD tissues and normal lung tissues were then identified. Based upon the above genes, three distinct immune patterns in the TCGA-LUAD cohort were identified. The ssGSEA and CIBERSORT were applied for immune profiling and characterization of different subtypes. A four-gene prognostic signature for LUAD patients was established based on the DEGs between the subtypes using stepwise multi-Cox regression. TCGA-LUAD cohort was used as training set. Five GEO-LUAD datasets and an independent cohort containing 112 LUAD samples were used for validation. TIDE (tumor immune dysfunction and exclusion) and drug sensitivity analyses were also performed.ResultsMacrophage-related differentially expressed genes were found out using the publicly available scRNA-seq data of LUAD. Three different immune patterns which were proved to have distinct immune infiltration characteristics in the TCGA-LUAD cohort were recognized based on the above macrophage-related genes. Thereafter, 174 DEGs among the above three different immune patterns were figured out; on the basis of this, a four-gene prognostic signature was constructed. This signature distinguished the prognosis of LUAD patients well in various GSE datasets as well as our independent cohort. Further analyses revealed that patients which had a higher risk score also accompanied with a lower immune infiltration level and a worse response to several immunotherapy biomarkers.ConclusionThis study highlighted that macrophage were significantly associated with TME diversity and complexity. The four-gene prognostic signature could be used for predicting outcomes and immune landscapes for patients with LUAD.