Peroxidase-induced enhancement of chemiluminescence by murine peritoneal macrophages

A number of substances have been shown to enhance the respiratory burst (RB) of macrophages. Many of these substances are not normally found in vivo. The present study suggests that a group of enzymes characterized as peroxidases have the ability to significantly enhance the RB and concomitant phagocytosis by murine peritoneal macrophages. Horseradish peroxidase (HRP), lactoperoxidase (LPO), and microperoxidase (MPO) can significantly augment these functions. Both resident and thioglycollate-induced macrophages exhibited enhanced chemiluminescence (CL) upon exposure to HRP, however, the effect was more pronounced with the latter. The increase in CL was correlated with an increase in production of superoxide, which was measured by reduction of cytochrome c. Horseradish peroxidase immobilized on an inert carrier, was capable of enhancing the RB suggesting that it does not have to enter the cell in order to function. Hemin, hematoheme and hematoporphyrin had little effect on macrophage stimulated CL. All of the peroxidases tested caused increased phagocytosis of opsonized zymosan. These studies indicate that peroxidases are capable of stimulating the RB, phagocytosis and possibly other macrophage functions.     

Regulation of RAW264 macrophage morphology and spreading: studies with protein kinase C activators, inhibitors and a cyclic AMP analog

Protein kinases C and A probably play important roles in membrane signal transduction. To test the role of protein kinases in macrophage spreading, we have measured cell perimeters in the absence and presence of protein kinase C activators, inhibitors and a cAMP analog. Scanning electron microscopy indicated that macrophages spread extensively in the presence of protein kinase C activators. In contrast, protein kinase C inhibitor and dbcAMP (N6-2'-O-di-butyryladenosine 3':5'-cyclic monophosphate AMP) promote a round cell morphology with many surface folds. Quantitative optical microscopy experiments showed that the maximal effects of these reagents were achieved within 30 min. The protein kinase C activators dioctonylglycerol (3 microM), phenylephrine (1 microM), and phorbol myristate acetate (1 micrograms/ml) increased macrophage spreading. Similarly, the calcium ionophore A23187 (1 microgram) increased spreading. In contrast, the protein kinase C inhibitors chlorpromazine (30 microM), sphingosine (10 microM), trifluoroperazine (10 microM), and H-7 (10 microM) significantly reduce macrophage spreading. The analog dibutyryl cAMP (30 microM) abrogates the effects of protein kinase C activators. These data suggest that protein kinase C participates in the regulation of macrophage spreading. Furthermore, the protein kinase A activator dibutyryl cAMP can inhibit the effects of protein kinase C activators.     

Disruption of filamentous actin inhibits human macrophage fusion.

The foreign body reaction to implanted biomaterials, characterized by the presence of macrophages and foreign body giant cells (FBGC), can result in structural and functional failure of the implant. Recently, we have shown that interleukin-4 and interleukin-13 can independently induce human macrophage fusion to form FBGC via a macrophage mannose receptor (MR) -mediated pathway. The MR is believed to mediate both endocytosis of glycoproteins and phagocytosis of microorganisms, which bear terminal mannose, fucose, N-acetylglucosamine, or glucose residues. Polarization of microfilaments to closely apposed macrophage membranes as observed with fluorescence confocal microscopy led us to ask whether MR-mediated fusion occurred via a filamentous actin-dependent pathway. Cytochalasins B and D and latrunculin-A, agents that disrupt microfilaments, inhibited macrophage fusion in a concentration-dependent manner. The concentrations of cytochalasins D and B that inhibited fusion did not significantly decrease macrophage adhesion, spreading, or motility but did inhibit internalization of Candida albicans during interleukin-13-enhanced, MR-mediated phagocytosis. Very low concentrations of cytochalasin B (< 2 microM) induced a slight enhancement of macrophage fusion. Taken together, the results of this study suggest that cytokine-induced, MR-mediated macrophage fusion requires an intact F-actin cytoskeleton and that the mechanism of fusion is similar to phagocytosis.--DeFife, K. M., Jenney, C. R., Colton, E., Anderson, J. M. Disruption of filamentous actin inhibits human macrophage fusion.     

Activation of macrophages by peroxidases

Peritoneal macrophages from C57BL/6 mice were activated in vitro with various peroxidases and their cytotoxic activity toward 3T12 cells was determined. Destruction of 3T12 cells by macrophages stimulated with horseradish peroxidase, lactoperoxidase, and microperoxidase was observed at peroxidase concentrations as low as 9, 1.6, and 200 nM, respectively. A 50% cytotoxic effect was obtained at peroxidase concentrations of 0.9, 1.6, and 1.5 microM, respectively. The macrophage-stimulating activity of horseradish peroxidase was not destroyed by boiling. This, together with the high activity of microperoxidase, indicates that the macrophage-stimulating activity of the peroxidases is probably associated with the heme portion of the enzymes. On a molar basis the peroxidases are much less potent macrophage activators than interferon (alpha + beta) and endotoxin. Nevertheless, our data clearly indicate that peroxidases are a group of enzymes capable of inducing macrophage activation, resulting in cytostatic and/or cytocidal activity.     

Kojic acid, a secondary metabolite from Aspergillus sp., acts as an inducer of macrophage activation

KA (kojic acid) is a secondary metabolite isolated from Aspergillus fungi that has demonstrated skin whitening, antioxidant and antitumour properties among others. However, limited information is available regarding its effects on macrophages, the major cell involved in cell defence. The aim of the present study was to analyse whether KA affects functional properties related to macrophage activation, such as phagocytosis and spreading ability over a substrate. Treatment of resident macrophages with 50 μg/ml KA for 1 h induced both morphological and physiological alterations in cells. Immunofluorescence microscopy revealed enhanced cell spreading and an increase in cell surface exposure, associated with a rearrangement of microtubules, actin filaments and intermediate filaments. KA also potentiated phagocytosis by macrophages, as demonstrated by the increase in phagocytic activity towards yeast, when compared to untreated cells. KA increased the production of ROS (reactive oxygen species), but not NO (nitric oxide) production. Three tests were used to assess cell viability; MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], NR (neutral red) uptake and PI (propidium iodide) exclusion test, which showed that macrophages maintain their viability following KA treatment. Results indicate that KA can modulate macrophage activation through cytoskeleton rearrangement, increase cell surface exposure, enhance the phagocytic process and ROS production. The study demonstrates a new role for KA as a macrophage activator.     

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.     

Actin polymerization in macrophages in response to oxidized LDL and apoptotic cells: role of 12/15-lipoxygenase and phosphoinositide 3-kinase

Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages. However, unlike apoptotic cells, OxLDL did not cause specific translocation of 12/15-LO to the cell membrane, neither in macrophages nor in GFP-15LO-transfected COS-7 cells. Moreover, inhibition of 12/15-LO activity in macrophages by a specific inhibitor or by 12/15-LO gene disruption did not affect OxLDL-induced actin polymerization. Among LDL modifications modeling OxLDL, LDL modified by incubation with 15LO-overexpressing fibroblasts was as active in eliciting F-actin response as was OxLDL. This LDL modification is well known to produce minimally modified LDL (mmLDL), which is bioactive and carries lipid oxidation products similar to those produced by 12/15-LO catalysis. MmLDL activated phosphoinositide 3-kinase (PI3K), and PI3K inhibitors abolished mmLDL-induced macrophage spreading. We hypothesize that OxLDL and mmLDL may contribute oxidized lipids to the macrophage cell membrane and thereby mimic intracellular 12/15-LO activity, which leads to uncontrolled actin polymerization and dramatic cytoskeletal changes in macrophages.     

Fc-receptor-mediated phagocytosis occurs in macrophages without an increase in average [Ca++]i

The calcium ion has been implicated as a cytosolic signal or regulator in phagocytosis. Using the Ca++-sensitive photoprotein aequorin we have measured intracellular free Ca++ ion concentration ([Ca++]i) in thioglycolate-elicited mouse peritoneal macrophages during phagocytosis and IgG-induced spreading. Macrophages plated on glass were loaded with aequorin and [Ca++]i was then measured from cell populations, both as previously described (McNeil, P. L., and D. L. Taylor, 1985, Cell Calcium, 6:83-92). Aequorin indicated a resting [Ca++]i in adherent macrophages of 84 nM and was responsive to changes in [Ca++]i induced by the addition of Mg-ATP (0.1 mM) or serum to medium. However, during the 15 min required for phagocytosis of seven or eight IgG-coated erythrocytes per macrophage loaded with aequorin, we measured no change in [Ca++]i. Similarly, the ligation of Fc-receptors that occurs when macrophages spread on immune complex-coated coverslips did not change macrophage [Ca++]i. In contrast, a rise in [Ca++]i of macrophages was measured during phagocytosis occurring in a serum-free saline of pH 7.85, and as a consequence of incubation with quin2 A/M. We estimate that had a change in [Ca++]i occurred during phagocytosis, aequorin would have detected a rise from 0.1 to 1.0 microM taking place in as little as 2% of the macrophage's cytoplasmic volume. We therefore suggest that either Ca++ is not involved as a cytoplasmic signal for phagocytosis or that increases in [Ca++]i during phagocytosis are confined to such small regions of cytoplasm as to be below the limits of detection by our cellular averaging method. Our data emphasizes, moreover, the need for well-defined, nonperturbing conditions in such measurements of [Ca++]i.     

Poly(I):poly(C)-enhanced alveolar and peritoneal macrophage phagocytosis: quantification by a new method utilizing fluorescent beads

Phagocytosis is an important immune function to quantify. This immune response may be modulated by exposure to biological response modifiers or by exposure to pollutants. A new technique for quantifying nonspecific phagocytosis of alveolar and peritoneal macrophages in the same animal has been developed that utilizes fluorescent polystyrene beads. When incorporated into inhalation studies, this technique can be used to determine whether the toxic effect of an inhaled pollutant is local (effect on alveolar macrophages), systemic (effect on peritoneal macrophages), or both local and systemic. This method results in a determination of both the level of phagocytosis (the percentage of phagocytic macrophages) and the macrophage specific activity (the number of beads phagocytized per macrophage). This method also allows a determination of adherence by quantifying the number of particles in contact with, but not phagocytized by, the macrophage. Macrophage preparations were incubated with fluorescent beads for 2 hr and cyto-centrifuged onto a glass slide. Fluorescent beads present on the slide or cell-associated but not ingested by phagocytosis were removed by immersing the slide containing the macrophage preparation in methylene chloride for 15-30 sec. Fluorescent beads ingested by phagocytosis were then easily quantified with a fluorescence microscope. This technique was used to assess the baseline levels of phagocytosis for rat alveolar and peritoneal macrophages from the same animal and the kinetics and level of enhanced phagocytosis for alveolar and peritoneal macrophages after injection with the interferon inducer polyinosinate-polycytidylate (poly(I):poly(C)). The kinetics of enhanced alveolar and peritoneal macrophage phagocytosis by poly(I):poly(C) were similar; however, stimulated phagocytic levels of peritoneal macrophages never reached the phagocytic activity observed for the resident, highly phagocytic alveolar macrophages. This elevated phagocytic activity is most likely due to interferon stimulated by particulate matter in the large volume of air processed by the lungs and is important for host defense against a number of different inhaled microorganisms.     

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.     

Opposite effects of bacterial lipopolysaccharide on Fc-receptor-mediated phagocytosis of two bone marrow-derived macrophage cell lines, BDM-1 and BDM-1W3.

We have reported the isolation and characterization of three factor-dependent macrophage cell lines from bone marrow cells of C3H/HeN mice. We have since isolated a subclone, BDM-1W3, from one of these cell lines. We found previously that BDM-1W3 has a different sensitivity to bacterial lipopolysaccharide (LPS) for growth than its parental cell line, BDM-1. In this report, we show that LPS inhibits BDM-1W3 phagocytosis of antibody-coated sheep erythrocytes (Fc-mediated phagocytosis), whereas it enhanced Fc-mediated phagocytosis by BDM-1. It was observed that a loss of Fc-receptor capacity parallels a loss of phagocytic activity in LPS-treated BDM-1W3 cells. LPS stimulated phagocytosis of latex beads by BDM-1 and BDM-1W3, suggesting that Fc-mediated phagocytosis and phagocytosis of latex beads differ in their regulatory mechanisms. When BDM-1 cells were cultured with LPS, they underwent drastic morphological changes, whereas LPS-treated BDM-1W3 cells did not change significantly. Gamma interferon enhanced FC-mediated phagocytosis by BDM-1, while it has no significant effect on that by BDM-1W3. These cell lines should be useful for studying signal transduction mechanisms in LPS-mediated macrophage activation.     

In Vitro Induction of Antibody-Dependent Cytotoxic Macrophages by the Local Anesthetic Lidocaine

Normal macrophages were activated to antibody-dependent cytotoxic effector cells by in vitro treatment with the local anesthetic lidocaine. Experiments on the dose-response and time course of the effect of lidocaine showed that incubation of normal macrophages with 10 mM lidocaine for 10 min at 28 C was enough for induction of antibody-dependent cellular cytotoxicity. The activation by lidocaine was accompanied by enhanced phagocytosis of sheep red blood cells (SRBC) sensitized with anti-SRBC antiserum, but not enhanced ingestion of polystyrene latex particles (PLP). These findings suggest that lidocaine, which has various effects on cell membranes, induces some perturbation of macrophage membranes, resulting in activation of Fc receptor functions in antibody-dependent cytotoxicity and phagocytosis.     

C57BL/6 and BALB/c bronchoalveolar macrophages respond differently to exercise.

Macrophages from prototypical Th1 strains (e.g., C57BL/6) and Th2 strains (e.g., BALB/c) are classified as M-1 and M-2 phenotypes. We investigated the different phagocytic responses between M-1 and M-2 bronchoalveolar macrophages (BAMs) under resting and two various exercise conditions. At rest, M-1 BAMs showed higher phagocytic capacity of unopsonized particles, higher expression of MARCO (macrophage receptor with collagenous structure), and higher generation of NO than M-2 BAMs. Severe exercise, but not moderate exercise, significantly enhanced both phagocytosis of unopsonized particles and expression of MARCO in M-2 BAMs. In contrast, M-1 BAMs were unaffected by either exercise protocol. The phagocytosis of unopsonized particles was largely mediated by MARCO, especially in M-1 BAMs. Secreted products from cultured M-2 BAMs isolated after severe exercise, but not those from M-1 BAMs, enhanced BAM phagocytosis. The cultured M-1 BAMs secreted phagocytosis inhibitors, and this effect could be blocked by NO antagonists. Moreover, the extent of phagocytosis suppression induced by M-1 BAM-secreted products correlated with their production of nitrite/nitrate. Exogenous NO donors as well as NO derivatives, nitrite and nitrate, suppressed the BAM phagocytosis. We propose that while the severe exercise-enhanced phagocytosis in M-2 BAMs was largely mediated by MARCO up-regulation and secretion of stimulators, the lack of exercise effect in M-1 BAMs could be partially due to the constitutive secretion of NO-related suppressors. In conclusion, genetically different mice use different strategies in regulating BAM activity under resting conditions and in response to various exercise paradigms.     

Single pulses of cytoplasmic calcium associated with phagocytosis of individual zymosan particles by macrophages

We have measured cytosolic free calcium levels ([Ca++]i) in individual macrophages during the phagocytosis of single zymosan particles. We report here that the contact of a macrophage with zymosan results in a rapid transient elevation of [Ca++]i. Each [Ca++]i pulse is symmetrical lasting for up to 30 seconds. In contrast, macrophage spreading is associated with repetitive [Ca++]i spiking occurring in salvos of up to four smaller spikes, each lasting for between 8 and 18 seconds. These qualitative and kinetic differences might suggest that the role of [Ca++]i in phagocytosis is distinct from its role in spreading.     

Differential binding to dorsal and ventral cell surfaces of fibroblasts: effect on collagen phagocytosis

Matrix remodeling by phagocytic fibroblasts is essential for growth and development but the regulatory processes are undefined. We evaluated the impact of spreading on the binding step of collagen phagocytosis with a novel culture system that more closely replicates phagocytosis in vivo than previous models. 3T3 cells were plated on collagen-coated beads, thereby loading only ventral surfaces (adhesion with spreading), or were allowed to spread on collagen films and then loaded with beads on their dorsal surfaces (adhesion without spreading). Ventral surfaces bound three-fold more beads than dorsal surfaces which was accompanied by accelerated phagosomal maturation. Arp3 and cortactin, markers of the actin-associated spreading machinery, strongly accumulated around ventrally but not dorsally loaded beads, suggesting that spreading contributes to enhanced binding of ventral surfaces. Further, ventral surfaces exhibited two-fold more free alpha2beta1 integrins, the major collagen receptors. Notably, compared to cells spread on collagen substrates, spreading cells exhibited a three-fold higher alpha2beta1 mobile fraction which was correlated with limited engagement of ventral receptors by actin filaments. Thus integrin ligation by actin filaments regulates the mobility of collagen receptors which in turn mediates the enhanced binding of collagen beads on spreading surfaces.     

Pulmonary surfactant protein A up-regulates activity of the mannose receptor,a pattern recognition receptor expressed on human macrophages

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that "shape" AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A(-/-) mice have reduced MR expression relative to SP-A(+/+). SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.     

Low intensity pulsed ultrasound accelerates macrophage phagocytosis by a pathway that requires actin polymerization, Rho, and Src/MAPKs activity

Phagocytosis is an essential event in the complex process of tissue repair. Here we examined the effect of low intensity pulsed ultrasound (US), which promotes fracture and wound healing, on phagocytosis by mouse macrophage cell line J774A.1 and human monocyte-derived macrophages. First, 10 to 40 min low intensity pulsed US increased uptake of serum opsonized E. coli by J774A.1 cells during a 50 min phagocytosis period. In addition, when the E. coli exposure time was varied between 35 to 80 min, the maximum increase in phagocytosis was observed in the first 35 min upon US exposure. In parallel, US induced robust actin polymerization in a time dependent manner in J774A.1 cells, showing the peak effect 30 min after stimulation. Interestingly, a low concentration of cytochalasin D (0.25-0.5 microM) prevented US-induced phagocytosis of E. coli. Furthermore, we demonstrated US enhanced activation of RhoA. Blocking its downstream effector Rho associated kinase (ROCK) with Y27632 abrogated US-induced phagocytosis. We also show that US induced activation of ERK and p38 MAPK. Pretreatment of the cells with the corresponding inhibitors PD98059 and SB203580 reduced US-induced phagocytosis. In addition, activity of tyrosine kinase Src was required for US-induced phagocytosis. Here Src represents an upstream activator of ERK and p38 MAPK. Depolymerization of actin by cytochalasin D prevented US-induced Src, ERK, and p38 activation. Our data provide a new insight into the cellular and molecular mechanisms by which low intensity pulsed US promotes tissue repair.     

Effects of cadmium and copper on antioxidant capacities, lignification and auxin degradation in leaves of pea (Pisum sativum L.) seedlings

Twelve-day-old seedlings of pea were treated for four days by 20 and 100 microM of Cd(NO3)2 or CuSO4. In leaves, all treatments caused an increase in the lipoperoxidation product rate. However, 20 microM of Cu did not affect the growth. Moreover, except for 20 microM of Cu, the activity of unspecific peroxidases, used as stress marker, was enhanced in cell walls of metal-stressed plants. No change in the antioxidant capacities was observed in plants treated with 20 microM of metal. At this dose, the Cd-reduced growth could be associated to an elevation in the activities of IAA oxidase and of lignifying peroxidases. Increase of these latter, in concert with loss in antioxidant capacities, would be responsible for the growth diminution after exposure to 100 microM of metal. However, the activity of lignifying enzymes was not affected by 100 microM of Cu. The contribution of cell fractions to enzymatic responses to stress is emphasized.     

Minimally modified LDL binds to CD14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells

Minimally modified low density lipoprotein (mmLDL) is a pro-inflammatory and pro-atherogenic lipoprotein that, unlike profoundly oxidized LDL (OxLDL), is not recognized by scavenger receptors and thus does not have enhanced uptake by macrophages. However, here we demonstrate that mmLDL (as well as OxLDL) induces actin polymerization and spreading of macrophages, which results in such pro-atherogenic consequences as inhibition of phagocytosis of apoptotic cells but enhancement of OxLDL uptake. We also demonstrate for the first time that the lipopolysaccharide receptor, CD14, and toll-like receptor-4/MD-2 are involved in these mmLDL effects. Macrophages of the J774 cell line exhibited higher mmLDL binding and F-actin response than its CD14-deficient mutant, LR-9 cells. Similarly, Chinese hamster ovary cells transfected with human CD14 specifically bound mmLDL and responded with higher F-actin compared with control cells. Macrophages from C3H/HeJ mice, which have a point mutation in the Tlr4 gene, responded with lower F-actin to mmLDL and did not spread as well as macrophages from control animals. A significantly higher F-actin response was also observed in Chinese hamster ovary cells transfected with human toll-like receptor-4/MD-2 but not with TLR4 alone or TLR2. Thus, in addition to inhibition of phagocytosis, the recognition of mmLDL by macrophage lipopolysaccharide receptors results in convergence of cellular immune responses to products of microorganisms and to oxidation-specific self-antigens, which could both influence macrophage function and atherogenesis.     

Enhancement of macrophage Fc-dependent phagocytosis by resident thymocytes: effect of a unique heat-stable lymphokine

Lymphocytes, activated by lectins or specific antigens, have been shown to enhance macrophage phagocytosis through the elaboration of a heat-labile soluble factor(s). Recent evidence from our laboratory revealed that resident (nonactivated) murine thymocytes and splenic lymphocytes increase peritoneal macrophage glucose metabolism through the elaboration of a heat-stable soluble factor(s). Therefore, we investigated the effect of resident lymphocyte subpopulations on macrophage Fc-dependent phagocytosis. Thioglycollate-elicited and resident peritoneal macrophages from BALB/c mice were cultured in serum-free media with syngeneic resident thymocytes or splenic T lymphocytes. Macrophage Fc-dependent phagocytosis was assayed by measuring the ingestion of 51CrSHEA. After 4 days in vitro, resident thymocytes produced a mean 160 (+/- 31) and 136% (+/- 22) increase in Fc-dependent phagocytosis by thioglycollate-elicited (thio-macrophages) and resident peritoneal macrophages, respectively. Splenic T lymphocytes increased thio-macrophage phagocytosis by 112% (+/- 41) under similar conditions. Macrophage Fc-dependent phagocytosis was increased after 24 hr of co-culture by supernatant derived from resident thymocytes and could be further enhanced by supernatant from Con A-activated thymocytes. Supernatant from guinea pig embryo fibroblasts did not increase macrophage phagocytosis. The soluble factor(s) was produced by resident thymocytes after 24 hr of preculture. This factor was active despite heating at 100 degrees C for 30 min whereas the effect of Con A-activated thymocyte supernatant was heat-labile. The stimulatory effect of resident thymocyte supernatant was not observed when the macrophages and supernatant were cultured in 2% FCS. In contrast to the factor(s) produced by resident thymocytes, the factor(s) in FCS that increased phagocytosis was heat-labile. These data suggest thymocytes and splenic T lymphocytes promote macrophage Fc-dependent phagocytosis in the absence of antigenic or lectin stimulation. This previously unrecognized effect of resident thymocytes is due to a unique heat-stable soluble factor(s) that is concealed in the presence of serum.