Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases

Mononuclear phagocytes have the capacity to directly participate in extracellular matrix turnover via secretion of neutral proteinases. We have studied the effects of in vivo and in vitro differentiation upon cellular content or secretion of a spectrum of neutral proteinases, along with a counter-regulatory metalloproteinase inhibitor (TIMP). We found 1) matrix-degradative serine proteinases (leukocyte elastase and cathepsin G) were lost during cellular maturation and/or differentiation; 2) the 92-kDa type IV/type V collagenase and TIMP were secreted earliest in mononuclear phagocyte differentiation, whereas stromelysin secretion was observed only by LPS-stimulated alveolar macrophages; 3) exposure of alveolar macrophages, but not monocytes, to phorbol esters and LPS resulted in markedly augmented secretion of all studied metalloproteinases and TIMP; 4) monocyte-derived macrophages partially (but not completely) mimicked the metalloproteinase secretory phenotype of alveolar macrophages; and 5) the secretory phenotype of alveolar macrophages for interstitial collagenase (but not TIMP) was largely lost during in vitro culture. These results underscore the complexity of the process of differentiation in human mononuclear phagocytes, and provide insights into the variable capacity of mononuclear phagocytes to degrade extracellular matrix components. Moreover, we anticipate that human mononuclear phagocytes at various stages of differentiation will provide a useful model system for study of the variable regulation of secretion of human matrix-degrading metalloproteinases.     

Production of multilineage growth factors by hematopoietic stromal cells: an intercellular regulatory network involving mononuclear phagocytes and interleukin-1

In the past 8 years, our group has carried out a series of in-vitro studies designed to characterize the role of mononuclear phagocytes as regulators of human hematopoiesis. The results of this program of investigation, some of which are reviewed below, led to the discovery that mononuclear phagocytes are more efficient recruitors of growth factor release by other cells than they are direct stimulators of progenitor cell growth. Specifically, mononuclear phagocytes release soluble factors (MRA) that stimulate other cells, including vascular endothelial cells, skin fibroblasts, and marrow fibroblasts, to release multilineage hematopoietic growth factors. Experiments designed to purify and characterize these monokines indicated unambiguously that the MRA that stimulates granulocyte/macrophage colony stimulating factor (GM-CSF) release is interleukin-1 (IL-1). Based on these observations and recent observations by other groups on the hematopoietic effects of other monokines including tumor necrosis factor alpha, we argue that mononuclear phagocytes serve as important regulators of hematopoiesis by producing monokines that, in turn, induce the expression of multiple hematopoietic growth factor genes in stromal cells of the hematopoietic microenvironment. Because IL-1 molecules and the mononuclear phagocytes producing them are evolutionarily conserved, and in view of the heterogeneous nonhematopoietic effects of these monokines, studies on their role in hematopoiesis may also provide new understanding of the molecular evolution of multicellular organisms.     

1-0-Hexadecyl-2-acetyl-sn-glycerol stimulates differentiation of HL-60 human promyelocytic leukemia cells to macrophage-like cells

The human promyelocytic leukemia cell line HL-60 can be differentiated to cells resembling either neutrophils or mononuclear phagocytes by a diverse group of stimuli. However, the underlying mechanisms remain unknown. We report that 1-0-hexadecyl-2-acetyl-sn-glycerol inhibits the growth of HL-60 cells and induces differentiation to cells resembling mononuclear phagocytes. HL-60 cultures incubated for 6 days with 1-0-hexadecyl-2-acetyl-sn-glycerol (5 micrograms/ml) demonstrated a ten-fold increase in nonspecific esterase activity, and produced cells with morphological features similar to those of monocytes and macrophages. Higher concentrations of 1-0-hexadecyl-2-acetyl-sn-glycerol significantly inhibited the growth of HL-60 cells and resulted in the virtual absence of cells resembling the original HL-60 line. 1-0-Oleoyl-2-acetyl-rac-glycerol added under the same conditions did not induce cell differentiation or inhibit cell growth.     

Role of vitamin D in bone resorption.

The idea that vitamin D must function at the bone site to promote bone mineralization has long existed since its discovery as an anti-rachitic agent. However, the definite evidence for this is still lacking. In contrast, much evidence has accumulated that 1 alpha,25(OH)2D3 in involved in bone resorption. 1 alpha,25(OH)2D3 tightly regulates differentiation of osteoclast progenitors into osteoclasts. Osteoclast progenitors have been thought to belong to the monocyte-macrophage lineage. 1 alpha,25(OH)2D3 greatly stimulates differentiation and activation of mononuclear phagocytes. Recent reports have indicated that differentiation of mononuclear phagocytes into osteoclasts is strictly regulated by osteoblastic cells, the process of which is also stimulated by 1 alpha,25(OH)2D3. In the differentiation of mononuclear phagocytes into osteoclasts, the target cells for 1 alpha,25(OH)2D3 appear to be osteoblastic stromal cells. Osteoblastic cells produce several proteins such as BGP, MGP, osteopontin and the third component of complement (C3) in response to the vitamin. They appear to be somehow involved in osteoclast differentiation and functions. Thus, 1 alpha,25(OH)2D3 seems to be involved in the differentiation of osteoclast progenitors into osteoclasts directly and also by an indirect mechanism involving osteoblastic cells. The precise role of osteoblastic cells in osteoclast development has to be elucidated in the future.     

CX3CR1+ lung mononuclear phagocytes spatially confined to the interstitium produce TNF-α and IL-6 and promote cigarette smoke-induced emphysema

Increased numbers of macrophages are found in the lungs of smokers and those with chronic obstructive pulmonary disease. Experimental evidence shows the central role of macrophages in elaboration of inflammatory mediators such as TNF-α and the progression toward cigarette smoke-induced emphysema. We investigated the role of CX3CR1 in recruitment of mononuclear phagocytes, inflammatory cytokine responses, and tissue destruction in the lungs after cigarette smoke exposure. Using mice in which egfp is expressed at the locus of the cx3cr1 gene, we show that alveolar macrophages increased transmembrane ligand CX3CL1 expression and soluble CX3CL1 was detectable in the airspaces, but cx3cr1(GFP/GFP) and cx3cr1(GFP/+) mice failed to show recruitment of CX3CR1(+) cells into the airspaces with cigarette smoke. In contrast, cigarette smoke increased the accumulation of CX3CR1(+)CD11b(+) mononuclear phagocytes that were spatially confined to the lung interstitium and heterogenous in their expression of CD11c, MHC class II, and autofluorescent property. Although an intact CX3CL1-CX3CR1 pathway amplified the percentage of CX3CR1(+)CD11b(+) mononuclear phagocytes in the lungs, it was not essential for recruitment. Rather, functional CX3CR1 was required for a subset of tissue-bound mononuclear phagocytes to produce TNF-α and IL-6 in response to cigarette smoke, and the absence of functional CX3CR1 protected mice from developing tissue-destructive emphysema. Thus, CX3CR1(+) "tissue resident" mononuclear phagocytes initiate an innate immune response to cigarette smoke by producing TNF-α and IL-6 and are capable of promoting emphysema.     

Macrophages and fibrosis: How resident and infiltrating mononuclear phagocytes orchestrate all phases of tissue injury and repair

Certain macrophage phenotypes contribute to tissue fibrosis, but why? Tissues host resident mononuclear phagocytes for their support to maintain homeostasis. Upon injury the changing tissue microenvironment alters their phenotype and primes infiltrating monocytes toward pro-inflammatory macrophages. Several mechanisms contribute to their deactivation and macrophage priming toward anti-inflammatory and pro-regenerative macrophages that produce multiple cytokines that display immunosuppressive as well as pro-regeneratory effects, such as IL-10 and TGF-beta1. Insufficient parenchymal repair creates a tissue microenvironment that becomes dominated by multiple growth factors that promote the pro-fibrotic macrophage phenotype that itself produces large amounts of such growth factors that further support fibrogenesis. However, the contribution of resident mononuclear phagocytes to physiological extracellular matrix turnover implies also their fibrolytic effects in the late stage of tissue scaring. Fibrolytic macrophages break down fibrous tissue, but their phenotypic characteristics remain to be described in more detail. Together, macrophages contribute to tissue fibrosis because the changing tissue environments prime them to assist and orchestrate all phases of tissue injury and repair. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Could a B-1 Cell Derived Phagocyte “Be One” of the Peritoneal Macrophages during LPS-Driven Inflammation?

The inflammatory response is driven by signals that recruit and elicit immune cells to areas of tissue damage or infection. The concept of a mononuclear phagocyte system postulates that monocytes circulating in the bloodstream are recruited to inflamed tissues where they give rise to macrophages. A recent publication demonstrated that the large increase in the macrophages observed during infection was the result of the multiplication of these cells rather than the recruitment of blood monocytes. We demonstrated previously that B-1 cells undergo differentiation to acquire a mononuclear phagocyte phenotype in vitro (B-1CDP), and we propose that B-1 cells could be an alternative origin for peritoneal macrophages. A number of recent studies that describe the phagocytic and microbicidal activity of B-1 cells in vitro and in vivo support this hypothesis. Based on these findings, we further investigated the differentiation of B-1 cells into phagocytes in vivo in response to LPS-induced inflammation. Therefore, we investigated the role of B-1 cells in the composition of the peritoneal macrophage population after LPS stimulation using osteopetrotic mice, BALB/Xid mice and the depletion of monocytes/macrophages by clodronate treatment. We show that peritoneal macrophages appear in op/op((-/-)) mice after LPS stimulation and exhibit the same Ig gene rearrangement (VH11) that is often found in B-1 cells. These results strongly suggest that op/op((-/-)) peritoneal "macrophages" are B-1CDP. Similarly, the LPS-induced increase in the macrophage population was observed even following monocyte/macrophage depletion by clodronate. After monocyte/macrophage depletion by clodronate, LPS-elicited macrophages were observed in BALB/Xid mice only following the transfer of B-1 cells. Based on these data, we confirmed that B-1 cell differentiation into phagocytes also occurs in vivo. In conclusion, the results strongly suggest that B-1 cell derived phagocytes are a component of the LPS-elicited peritoneal macrophage population.     

Lineage specific receptors used to identify a growth factor for developmentally early hemopoietic cells: assay of hemopoietin-2

A new approach, based on the occurrence of receptors for the mononuclear phagocyte lineage specific hemopoietic growth factor (HGF) colony stimulating factor-1 (CSF-1) on developmentally early multipotent cells, is utilized to detect and assay rapidly another HGF, hemopoietin-2. This method is also used to determine the relative maturity of hemopoietin-2 target cells, to investigate synergism between hemopoietin-2 and CSF-1, and to measure CSF-1 receptor levels on maturing cells. While the target cell specificities of hemopoietin-2 and CSF-1 overlap, hemopoietin-2 causes the appearance of developmentally earlier 125I-CSF-1 binding cells de novo in the absence of CSF-1. Increased CSF-1 receptor densities are observed on cells incubated with either HGF, consistent with acquisition of the capacity for increased expression of the receptor by mononuclear phagocyte progenitor cells just prior to their differentiation to adherent mononuclear phagocytes. Together, both HGFs have a synergistic effect on the generation of 125I-CSF-1 binding cells with elevated CSF-1 receptor densities. Preliminary characterization of hemopoietin-2 from medium conditioned by WEHI-3 cells indicates that it is very similar to, if not identical with, interleukin-3 (IL-3) and the HGF(s) acting on multipotential cells and cells giving rise to erythroid cells, granulocytes, mononuclear phagocytes, and megakaryocytes. Purified IL-3 was shown to possess hemopoietin-2 activity.     

Macrophages in resistance to candidiasis.

Candida albicans, an increasingly common opportunistic pathogenic fungus, frequently causes disease in immunodeficient but not immunocompetent hosts. Clarifying the role of the phagocytic cells that participate in resistance to candidiasis not only is basic to understanding how the host copes with this dimorphic pathogen but also will expedite the development of innovative prophylactic and therapeutic approaches for treating the multiple clinical presentations that candidiasis encompasses. In this review, we present evidence that a diverse population of mononuclear phagocytes, in different states of activation and differentiation and from a variety of host species, can phagocytize C. albicans blastoconidia via an array of opsonic and nonopsonic mechanisms and can kill C. albicans blastoconidia and hyphae by means of oxygen-dependent and -independent mechanisms. Reactive nitrogen intermediates should now be added to the well-established candidacidal reactive oxygen intermediates of macrophages. Furthermore, what were thought to be two independent pathways, i.e., nitric oxide and superoxide anion, have now been shown to combine to form a potent macrophage candidacidal molecule, peroxynitrite. In contrast to monocytes and neutrophils, which are important in resistance to early stages of C. albicans infections, more differentiated macrophages activated by cytokines such as gamma interferon participate in the acquired resistance of hosts with C. albicans-specific, cell-mediated immunity. Evidence presented in this review demonstrates that mononuclear phagocytes, in some instances in the absence of other professional phagocytes such as neutrophils, play an import role in resistance to systemic and mucosal candidiasis.     

Quantitative immunocytochemical characterization of mononuclear phagocytes. I. Monoblasts, promonocytes, monocytes, and peritoneal and alveolar macrophages

The purpose of the present study was to compare the phenotype of tissue macrophages with that of their precursors in the bone marrow and blood. The phenotype was determined on the basis of the quantitative binding of monoclonal antibodies to cell-surface antigens (antigen F4/80, complement receptor III, Fc receptor II, Ia antigen, common leukocyte antigen, and Mac-2 and Mac-3 antigens) on individual mononuclear phagocytes. Monoclonal antibody binding to cells, detected by the biotin-avidin immunoperoxidase procedure, was quantitated by cytophotometric determination of the amount of enzyme reaction product on cells. The results of this quantitation are expressed as the median of the specific absorbance per unit of cell-surface area (0.25 micron2) and per cell. Shortly after collection of the mononuclear phagocytes, binding of all monoclonal antibodies except those directed against the common leukocyte and Mac-2 antigens to peritoneal macrophages was enhanced compared with binding to blood monocytes; for alveolar macrophages we found reduced binding of monoclonal antibodies F4/80 and M1/70 (complement receptor III) and enhanced binding of monoclonal antibodies with specificity for the common leukocyte antigen and Mac-2 and Mac-3 antigens. The results obtained with cultured mononuclear phagocytes show that during the development from monoblast to tissue macrophages, monoclonal antibody binding to the various types of mononuclear phagocyte, expressed per unit of cell-surface area, was not significantly altered except that of M3/38 (Mac-2 antigen) to peritoneal macrophages and that of F4/80 and M1/70 (complement receptor III) to alveolar macrophages. Expressed on a per cell basis, the results show an increase in the binding of all monoclonal antibodies except those directed against the Fc receptor II and Mac-3 antigen during the development from promonocytes to peritoneal macrophages; binding of most monoclonal antibodies to alveolar macrophages was considerably lower than that to blood monocytes. It is concluded that the expression of the various cell-surface antigens alters during mononuclear phagocyte differentiation. The expression changed also during culture, although distinct patterns of alteration could not be distinguished.     

Effects of differentiation in vivo and of lymphokine treatment in vitro on the mobility of C3 receptors of human and mouse mononuclear phagocytes

The C3 receptors of human peripheral blood monocytes are able to move laterally within the plasma membranes of the cells and remain mobile even when the cells develop into "macrophages" in vitro. In contrast, the C3 receptors of mouse peritoneal macrophages are immobile. To determine whether these differences are species differences or differences between cells of different stages of differentiation, we assessed the mobility of C3 receptors of mouse peripheral blood monocytes and of human pulmonary alveolar and peritoneal macrophages. The C3 receptors of mouse monocytes were mobile, whereas the C3 receptors of human tissue macrophages were immobile. The C3 receptors of macrophages mediate avid particle binding but do not normally promote ingestion. We have described a unique lymphokine that activates mouse peritoneal macrophage C3 receptors for phagocytosis by freeing them from their plasma membrane anchors. In the present experiments, we found that the lymphokine also freed the C3 receptors of human macrophages and activated them for phagocytosis. We conclude that the immobilization of C3 receptors appears to be a marker for the differentiation of human and mouse mononuclear phagocytes, that the differentiation of mononuclear phagocytes is influenced by the milieu in which the cells develop, that in vitro-differentiated macrophages may not accurately represent tissue macrophages, and that a lymphokine activates the C3 receptors of both human and mouse macrophages for phagocytosis by allowing the receptors lateral mobility within the cell plasma membrane.     

Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation

Dexamethasone, a synthetic glucocorticosteroid, was shown to modulate the colony-stimulating factor-dependent clonal growth of myeloid progenitor cells in semisolid agar cultures, enhancing the formation of granulocyte colonies (50–100%) and suppressing the formation of macrophage colonies (75–97%). Modulation of the pattern of myeloid colony formation by dexamethasone (12–125 nM) was brought about when the steroid was administered to 6-day cultures at the time of culture initiation and up to 72 hr later. Dexamethasone inhibited myeloid cell proliferation when administered to 5-day liquid cultures at culture initiation and up to 96 hr later. Dexamethasone (12–250 nM) also enhanced the phagocytic activity of bone marrow-derived mononuclear phagocytes toward heat-killed (HK) yeast cells (up to 100%) and IgG-coated sheep red blood cells (up to 60%). Enhancement of the phagocytic capability depended critically on the stage in culture at which dexamethasone was administered. Exposure to dexamethasone for 28 hr up to 96 hr of 96-hr cultures of bone marrow cells did not lead to a modulation of phagocytic activity of the developing mononuclear phagocytes. The presence of dexamethasone during the critical period of 96 hr to 120 hr after culture initiation led to an enhanced phagocytic capability, which was statistically significant already 12 hr after the administration of the glucocorticoid. Dexamethasone induced an enhanced phagocytic activity when administered at any time after culture initiation provided that it was in culture during this critical period. When added at 120 hr of culture, dexamethasone no longer enhanced the phagocytic capability of mononuclear phagocytes and when added later than 156 hr of culture suppressed it. Dexamethasone also suppressed (up to 68%) the phagocytic capability of resident and elicited peritoneal macrophages. The results suggest that glucocorticoids shift the balance of granulocyte vs. macrophage formation at early stages of precursor cell differentiation. Reduction in mononuclear phagocyte growth and enhancement of its phagocytic capability might reflect accelerated differentiation/maturation steps. The inhibitory effect of dexamethasone on macrophage formation and on the phagocytic capability of mature mononuclear phagocytes and peritoneal macrophages might be a relevant aspect of the in vivo immune suppression encountered after glucocorticoid administration.     

Macrophage growth inhibitors derived from the murine peritoneal cavity

Summary The murine peritoneal cavity contains factors that inhibit the in vitro growth and colony formation of macrophages. The inhibition of macrophage growth is not due to cell death. In the presence of inhibitors, the growth of colony-forming macrophages is suppressed, and small clusters are formed as a result of limited proliferation. The more mature mono-nuclear phagocytes (blood monocytes and peritoneal exudate macrophages) are more sensitive to the overall inhibitory effect of the peritoneal inhibitors than the less mature bone marrow mononuclear phagocytes. Furthermore, using dialysis and Amicon ultrafiltration, at least two inhibitors with differential inhibitory effects can be demonstrated. The colony formation of bone marrow mononuclear phagocytes is suppressed mainly by a protease-resistant, small molecular weight (<1,000) dialyzable inhibitor. In contrast, peritoneal exudate macrophages are sensitive to both the small molecular weight inhibitor and a protease-sensitive, large molecular weight (>12,000), nondialyzable inhibitor. The data suggest a possible existence of a dual inhibitor control on the proliferation of mononuclear phagocytes in vivo. In addition, the in vitro cultured peritoneal exudate cells are capable of producing inhibitors that mimic the activity of the in vivo inhibitors. This investigation was supported by Grants CA 09 11(SY) and AI15563(CCS) from the National Institutes of Health, Bethesda, MD     

Induction, maintenance, and reinduction of tumoricidal activity in bone-marrow-derived mononuclear phagocytes by macrophage-activating lymphokines

Rat bone-marrow-derived mononuclear phagocytes, induced to differentiate in vitro from precursors and virtually homogeneous with respect to the cell lineage, were the source of effector cells. These effector cells do not manifest spontaneous cytolytic activity in the resting state, but readily acquire marked long-term tumoricidal activity upon incubation with macrophage-activating lymphokines (MAF). MAF-induced tumoricidal activity of bone marrow-derived effector cells decays rapidly. However, in sharp contrast to tissue macrophages, bone marrow-derived mononuclear phagocytes retain in vitro responsiveness to a primary exposition to MAF over a period of several weeks, postcytolytic mononuclear phagocytes recover reactivity to MAF after a variable time interval.     

Non-glial phagocytes within the degenerating optic nerve of the newt (Triturus viridescens).

Two non-glial phagocytes were found to participate along with ependymoglial cells in Wallerian degeneration of the severed optic nerve of the newt (Triturus viridescens). The first type of non-glial cell (polymorphonuclear phagocyte) was positively identified as a neutrophil and participates in the early stages of degeneration. Cells of this type make a brief appearance, reaching a peak by the second postoperative day (2 p.o.d.), and quickly diminish until few can be found by 4 p.o.d. Neutrophils invade the degenerating optic nerve from surrounding connective tissue spaces, most likely, through channels which penetrate the nerve parenchyma. The second type of non-glial cell is an invading mononuclear phagocyte which exhibits characteristics of microglial cells reported in other vertebrate species. Such cells appear in the nerve much later than the neutrophils and towards the end of Wallerian degeneration (6-10 p.o.d.). Their mode of entry and exit appears to be the same as that reported for neutrophils. The neutrophils and microglial-like, mononuclear phagocytes may serve to supplement the histolytic action of the ependymoglial cells, picking up scattered fragments of degenerating myelin and axons.     

The role of phagocytes in the protective mechanisms of fish

Phagocytes are cells principally dedicated to the recognition and elimination of invading organisms and damaged tissue. Those described in fish are the granulocytes (particularly neutrophils) and mononuclear phagocytes (tissue macrophages and circulating monocytes). Their movement to sites of microbial invasion is an early event in the inflammatory response and the role of host-derived factors as attractants, such as eicosanoids, is discussed. Opsonins mediate the recognition between phagocyte and particle, and receptors for serum complement component C3 and the Fc fragment of opsonic antibody have been described. Fundamental to the protection offered by the phagocytes is their bactericidal larvacidal activity, which is closely associated with the production of oxygen free radicals. Phagocytes as accessory cells are discussed, including their role in antigen presentation. A knowledge of the modulation of phagocyte function, with activation by various substances and suppression by others, is important if protective responses are to be achieved by up-regulating phagocyte activity.     

Identification of osteoclasts and their differentiation from mononuclear phagocytes by enzyme histochemistry

Summary A double staining method is presented which allows the enzyme histochemical differentiation between osteoclasts (mono- and multinucleated forms) and mononuclear phagocytes (macrophages, multinucleated inflammatory giant cells). Osteoclasts are characterized by a strong acid phosphatase activity whereas macrophages and inflammatory giant cells show a variable non-specific esterase activity. The described method may be useful in studying the osteoclast origin and the extraosseus distribution of these cells.Supported by Deutsche Forschungsgemeinschaft, SFB 244,A1