Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha

Previously, we reported that exposure of bone marrow-derived macrophages (M phi) to a phagocytic stimulus in the simultaneous presence of interferon-gamma (IFN-gamma) induced these cells to generate nitrite (NO2-). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettii) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2- secretion. As shown here, the capacity of phagocytosis to elicit NO2- production by IFN-gamma-treated M phi was inhibited by antibody to murine recombinant tumor necrosis factor-alpha (rTNF-alpha), suggesting that phagocytosis enabled IFN-gamma to activate M phi via the induction of TNF-alpha as an autocrine second signal. M phi NO2- production in response to rIFN-gamma and either exogenous TNF-alpha or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism. However, addition of either Leishmania promastigotes or latex beads to M phi cultures simultaneously exposed to both IFN-gamma and exogenous murine or human rTNF-alpha further potentiated activation as measured by NO2- release. Furthermore, anti-TNF antibody failed to inhibit M phi responses to rIFN-gamma and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-alpha did not significantly affect NO2- production by IFN-gamma/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances M phi responses by a process more complex than the sole induction of TNF-alpha. Phagocytosis also increased M phi NO2- production elicited by IFN-gamma plus TNF-alpha in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating M phi microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.     

Modulation of alveolar macrophage phagocytosis by leukotrienes is Fc receptor-mediated and protein kinase C-dependent

We have previously established an important role for leukotrienes (LTs) in augmenting rat alveolar macrophage (AM) phagocytosis of Klebsiella pneumoniae opsonized with complete immune serum (IS), which contains the two well-known opsonins, immunoglobulin (Ig) G and complement (C). In this report, the specific opsonin requirements for LT modulation of AM phagocytosis and the dependence of this response on protein kinase (PK) C activity were investigated. Phagocytosis of K. pneumoniae opsonized with IS, non-immune serum, or heat-inactivated immune serum and of inert targets (IgG-opsonized fluorescent microspheres or C-opsonized sheep red blood cells) was examined. Inhibition of endogenous LT synthesis or action attenuated, whereas the addition of exogenous LTs augmented, phagocytosis only of targets opsonized with IgG. LTs had no effect on phagocytosis of C-opsonized or unopsonized targets. LTs did not affect adherence of IgG-opsonized targets, implying instead an enhancement of internalization. Macrophage internalization of phagocytic targets has previously been shown to require PKC activity. Pretreatment of AMs with the PKC inhibitors staurosporine or calphostin C, or with phorbol 12-myristate 13-acetate to deplete PKC, completely inhibited the ability of LTB(4) and largely inhibited the ability of LTC(4) to augment phagocytosis of IgG-opsonized microspheres. These results demonstrate that LT enhancement is confined to Fc receptor (FcR)-mediated phagocytosis. Moreover, PKC activation represents an important mechanism by which LTs promote FcR-mediated phagocytosis.     

Human macrophage function and physical exercise: phagocytic and histochemical studies

Summary Macrophages derived from human connective tissue were assayed for their enzyme content and phagocytic activity after physical exercise. A single exhaustive endurance-running test caused increased phagocytic and enzymatic activities of the macrophages. Thus, an exercise challenge activates the functional status of the cells. This effect of physical exercise on macrophages is inconsistent with the practical experience that high performance athletes suffer more frequently from harmless infectious diseases.     

Silica-induced apoptosis in murine macrophage: involvement of tumor necrosis factor-alpha and nuclear factor-kappaB activation

Although it is clearly established that surfactant protein A (SP-A) is secreted by type II pneumocytes as a component of pulmonary surfactant, its secretion pathway as well as its subcellular localization in the human lung are uncertain. We therefore studied the intracellular and intra-alveolar localization of SP-A in eight adult human lungs by immunohistochemistry and immunoelectron microscopy. Only type II pneumocytes could be identified as SP-A positive cells within the parenchymal region. SP-A was localized mainly in small vesicles and multivesicular bodies close to the apical plasma membrane. Only few lamellar bodies were weakly labeled at their outer membranes. Stereologic analysis showed this weak signal to be due to specific labeling. In the alveolar space, lamellar body-like surfactant forms in close proximity to tubular myelin were labeled for SP-A at their periphery. The strongest SP-A labeling was found over tubular myelin figures. Labeling for SP-A was also found in close association with the surface film and unilamellar vesicles. Our results support the hypothesis that, in the human lung, SP-A is mainly secreted into the alveolar space via an alternative pathway that largely bypasses the lamellar bodies. After secretion, the outer membranes of unwinding lamellar bodies become enriched with SP-A when tubular myelin formation is initiated. SP-A may also be involved in the transition of tubular myelin into the surface film.     

A phagocytic challenge with IgG-coated erythrocytes depresses macrophage respiratory burst and phagocytic function by different mechanisms

A phagocytic challenge with IgG-coated erythrocytes (EIgG) previously has been shown to cause impaired macrophage respiratory burst capacity and FcgammaR-mediated phagocytic function. Because both the respiratory burst and FcgammaR-mediated phagocytosis are dependent on the release of arachidonate (AA), we evaluated the effects of impaired AA release on the depression of macrophage function caused by a phagocytic challenge. Challenge with EIgG caused a depression of A23187-stimulated AA release that was associated with impaired phorbol myristate acetate (PMA)-stimulated H2O2 production and FcgammaR-mediated phagocytic function. In contrast, challenge with IgG-coated glass beads (BIgG) had no effect on either AA release or H2O2 production but did depress phagocytic function. Exogenous AA prevented the depression of H2O2 production but had no effect on phagocytic function. Phospholipase A2 (PLA2) activity was depressed under conditions where AA release was impaired. The depression of phagocytic function was correlated with a depression of both EIgG binding and FcgammaR expression. Thus, a phagocytic challenge with EIgG results in macrophage dysfunction by depressing PLA2 activity and depleting FcgammaR.     

Regulation of murine macrophage function by IL-4: IL-4 and IFN-gamma differentially regulate macrophage tumoricidal activation.

To understand the differential role of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the process of macrophage tumoricidal activation, we investigated the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide in activated murine macrophages and the effects of those lymphokines on prostaglandin E2 (PGE2)-mediated down-regulation. IFN-gamma and IL-4 increased lipopolysaccharide (LPS)-induced TNF-alpha production by different mechanisms because IL-4, unlike IFN-gamma, failed to overcome the LPS-hyporesponsiveness in C3H/HeJ mice. Moreover, only IFN-gamma synergized with LPS to induce nitric oxide production and blocked eicosanoid-mediated down-regulation. These differential effects of IFN-gamma and IL-4 on the select efferent cytolytic activities may be the result of an altered or different signal transduction pathway. Because potentiation of protein kinase C (PKC) activity by IFN-gamma has been previously documented, we next studied the role of IFN-gamma and IL-4 in alteration of enzymatic activity of PKC. Two lymphokines caused translocation of PKC from cytosol to membrane with different levels, providing a biochemical basis for explaining how two lymphokines lead to different phenotypic responses. Although treatment of macrophages with IFN-gamma and IL-4 gave rise to a similar enhancing effect on macrophage TNF-alpha production, these two lymphokines appeared to differentially regulate the overall functional state of macrophages for tumour cell killing capability. Additionally, this differential regulation seems to be accomplished in part by different biochemical events.     

Photometric microassay for quantitation of macrophage Fc and C3b receptor function

A photometric microassay has been developed to quantitate macrophage Fc and C3b receptor mediated binding and phagocytosis by measuring the absorbance of macrophage associated erythrocytes at 405 nm on an automated densitometer. The method compares favorably in sensitivity and kinetics to the 51Cr-labeled erythrocyte assay. Saturation and linear dose response kinetics were demonstrable for both total index and phagocytic index of either Fc receptor or C3b receptor. The assay allowed detection of significant differences in Fc receptor function with varying macrophage densities and between Fc receptor competent (C3HeB/FeJ) macrophages and Fc receptor deficient (C3H/HeJ) macrophages. A valid binding index was derived at 37 degrees C by computing the difference between the total and phagocytic indices, which compared favorably with binding studies at 4 degrees C. This new procedure provides a simple, rapid and reproducible microassay for the quantitation of Fc/C3b receptor dependent binding and phagocytosis which offers distinct advantages over the laborious rosette assay and the 51Cr-labeled erythrocyte assay.     

Effects of pretreatment with protein kinase C activators on macrophage activation for tumor cytotoxicity, secretion of tumor necrosis factor, and its mRNA expression

The effect of phorbol esters and mezerein pretreatment on macrophage (M phi) activation for tumor cytolysis, tumor necrosis factor (TNF) secretion, and TNF-alpha mRNA expression was investigated. Following pretreatment with various concentrations (0.01 to 10 micrograms/ml) of phorbol 12-myristate 13-acetate (PMA), phorbol-12,13-dibutyrate (PDBu), or mezerein for 16 h, murine peritoneal M phi were activated with M phi-activating factor (MAF) or calcium ionophore A23187 and tested for cytotoxicity in a 24-h cytolysis assay against 125-I-UdR-labeled P815 mastocytoma and NS-1 myeloma target cells. It was found that pretreatment with all three protein kinase C (PKc) activators inhibited M phi activation for cytotoxicity against P815 cells in a dose-dependent manner. Fifty percent inhibition was achieved at concentrations less than 0.1 micrograms/ml. The inhibition was partially reversible. In contrast, the pretreatment did not at all inhibit but significantly enhanced M phi activation for cytolysis against NS-1 cells. Furthermore, exposure to PMA augmented M phi activation by MAF and A23187 for TNF secretion upon stimulation with trace amounts of lipopolysaccharide (LPS). Although the pretreatment neither enhanced nor significantly reduced the synergistic effect of MAF and A23187 on TNF-alpha mRNA expression, it did increase the expression stimulated by LPS alone. Finally, the PKc activity in M phi treated with PMA, PDBu, and mezerein was down-regulated to about 10% of control. Taken together, our results suggest that: 1) PKc plays an important role in the transduction of activating signals for M phi activation by MAF and A23187 to mediate cytotoxicity against some (P815) but not other (NS-1) tumor cells, 2) the induction of TNF-alpha mRNA expression and TNF secretion may be achieved via a PKc-independent pathway, and 3) M phi are equipped with more than one signal transduction pathways for affecting distinct functional activities.     

A modified colorimetric assay of macrophage activation for intracellular cytotoxicity against Leishmania parasites

An in vitro method is described which colorimetrically assesses the activation of macrophages for intracellular cytotoxicity against the obligate intracellular parasite Leishmania donovani. The assay system uses a highly purified macrophage population derived from 10-day murine bone marrow cultures. These were infected in vitro as a suspension culture with viable L. donovani amastigotes and then exposed to activating agents. After 48 h the intracellular parasites were released by SDS lysis of the macrophages. Surviving Leishmania organisms were quantitated by their conversion of the chromophore MTT. The sensitivity of this method was comparable with the established method of [3H]dThd incorporation. This assay system has been used to show that there is a dual signal requirement (recombinant interferon-gamma and bacterial endotoxin (LPS] for effective activation of macrophages for leishmanicidal activity.     

Human peripheral blood monocyte and bronchoalveolar macrophage cytotoxicity for cultured human lung tumor cells

Human peripheral blood monocytes (PBM) and bronchoalveolar macrophages (BAM) were tested for cytotoxicity toward a cultured human lung tumor cell line, A549, using a 75Se-methionine post-labelling assay. Cytotoxicity of increasing numbers of PBM plateaud at an effector cell (E):target cell (T) ratio of 3:1. In contrast, BAM cytotoxicity was significantly lower than that of PBM at low E:T ratios but increased in a dose-dependent manner approaching 100% at an E:T ratio of 20:1, this increased cytotoxicity being due to cytolysis. PBM cytotoxicity appeared to be suppressed at least partly by a factor(s) liberated by PBM themselves. The different nature of the two effector cell populations' cytotoxic dose response curves and kinetic studies, and the inability of lipopolysaccharide to stimulate a level of PBM cytotoxicity attainable by BAM, suggested that the mechanism of cytotoxicity of the two cell populations differed or that BAM were more activated than PBM, or both.     

Microassay for photometric quantitation of macrophage mediated tumor cytotoxicity using an automated densitometer

A simple and reproducible microassay for the quantitation of macrophage mediated cytotoxicity is described. The method is based on the measurement of absorbance at 630 nm of residual Giemsa stained target cells and effector macrophages using an automated densitometer. Applying this novel method, it was possible to demonstrate time dependent growth characteristics of C3H/MCA and BHK/Py target cell lines. Using C3HeB/FeJ or C3H/HeJ murine effector macrophages and syngeneic transformed fibroblast target cells (C3H/MCA or 3T12), the method was further applied to demonstrate: (1) dose related activation of macrophages by lipopolysaccharide (LPS) and by macrophage activating factor (MAF); (2) synergistic augmentation of MAF-mediated macrophage cytotoxicity by LPS; (3) unresponsiveness of C3H/HeJ macrophages to LPS; and (4) increased cytotoxicity with increasing effector: target cell ratios. Guinea pig peritoneal macrophages were also shown to produce enhanced LPS or MAF-mediated cytotoxicity for C3H/MCA or BHK/Py target cells. The novel method was shown to compare favorably with results obtained by cytotoxic release of [3H]thymidine from prelabeled target cells. The advantages of the method are: (1) the elimination of the need for radioactive materials; (2) the ability to perform quantitation directly in microtiter plates; (3) the relative ease and rapidity in which experiments may be performed and quantitated; (4) its sensitivity and reproducibility; and (5) the ability to simultaneously quantitate and observe the biological events either microscopically or macroscopically.     

Type II pneumocyte-CD8+ T-cell interactions. Relationship between target cell cytotoxicity and activation

CD8+ T-cell responses play an important role in the clearance of respiratory virus infection, but may also contribute to lung injury in the process. The effector mechanisms involved in viral clearance and associated lung injury include both cytolytic and noncytolytic effector functions. Previously we have shown that CD8+ T-cell recognition of alveolar epithelial cells triggers chemokine expression by the epithelial cell and that this plays an important role in the inflammatory infiltration that ensues in the context of T cell-mediated injury (Zhao and colleagues, J. Clin. Invest. 2000;106:R49-R58). In the present study we sought to understand the relationship between alveolar cell cytotoxicity and chemokine expression, both of which occur as a result of CD8+ T-cell antigen recognition. Alveolar epithelial cells efficiently process and present overlapping viral epitopes, and CD8+ T-cell recognition of these class I major histocompatibility complex-restricted epitopes resulted in cytotoxicity of the alveolar cells by both wild-type and perforin-deficient T cells. However, the contribution of perforin-mediated lysis to the total cytotoxicity of alveolar cells by CD8+ T cells was minimal, and the majority of the lysis was attributable to tumor necrosis factor-alpha expressed by the T cell. CD8+ T-cell recognition also led to activation of nuclear factor-kappaB in the alveolar epithelial target cells, at levels inversely proportional to the effector/target (E:T) ratio. Finally, at varying E:T ratios, we demonstrated an inverse relationship between alveolar cell cytotoxicity and monocyte chemotactic protein-1 expression, both of which occur as a result of T-cell recognition. These findings may have important ramifications in understanding the relationship between viral clearance and lung injury.     

Characterization of murine macrophage Fc receptor-dependent phagocytosis and antibody-dependent cellular cytotoxicity during in vitro culture with interferons-gamma, alpha/beta and/or fetal bovine serum

Resident and oil-elicited inflammatory peritoneal macrophages (PM phi) from competent C3HeB/FeJ and genetically deficient C3H/HeJ mice were characterized for their Fc receptor (FcR)-dependent binding, phagocytic and ADCC functions during in vitro differentiation under the influence of mouse recombinant interferon-gamma (rIFN-gamma), interferon-alpha/beta (IFN-alpha/beta) fetal bovine serum (FBS), or in serum-free medium. Freshly cultured resident PM phi from C3HeB/FeJ mice had low levels of FcR-mediated phagocytosis in response to mouse monoclonal IgG gamma 2a, IgG gamma 2b or IgG gamma 1, opsonized sheep erythrocytes as compared to oil-elicited inflammatory PM phi from the same strain. Resident PM phi were uniformly upregulated in their FcR-dependent phagocytosis after 24-48 h in vitro culture with FBS to levels approximating that of freshly cultured inflammatory PM phi which were also further upregulated after 24 h in vitro culture with FBS. Both resident and inflammatory PM phi were upregulated largely by an autostimulatory process in that they increased their FcR-mediated phagocytosis in serum-free RPMI-1640 medium without the addition of rIFN-gamma or IFN-alpha/beta, although FBS further augmented FcR upregulation. A synergistic effect of FBS and rIFN-gamma was required for total reconstitution of FcR-mediated phagocytosis of FcR-incompetent C3H/HeJ inflammatory PM phi in that FBS or rIFN-gamma alone only partially reconstituted FcR function, whereas in combination full reconstitution occurred. Thus, macrophages from competent C3HeB/FeJ mice were upregulated in their FcR-mediated functions largely by an autostimulatory process, presumably dependent on endogenous of IFN-beta, whereas, genetically-deficient C3H/HeJ macrophages required exogenous rIFN-gamma in combination with fetal bovine serum for synergistic reconstitution of FcR functions. The uniform upregulation of FcR-dependent effector functions in vitro appears to provide an efficient system for enhanced immune function during differentiation which may be applicable to in vivo situations.     

Ginger extract inhibits LPS induced macrophage activation and function

Background  

Macrophages play a dual role in host defence. They act as the first line of defence by mounting an inflammatory response to antigen exposure and also act as antigen presenting cells and initiate the adaptive immune response. They are also the primary infiltrating cells at the site of inflammation. Inhibition of macrophage activation is one of the possible approaches towards modulating inflammation. Both conventional and alternative approaches are being studied in this regard. Ginger, an herbal product with broad anti inflammatory actions, is used as an alternative medicine in a number of inflammatory conditions like rheumatic disorders. In the present study we examined the effect of ginger extract on macrophage activation in the presence of LPS stimulation.     

Studies of macrophage function in murine systemic lupus erythematosus. 3. The nature, anatomical location, and reversibility of the phagocytic defect

The defect in phagocytosis and binding of antibody-coated sheep erythrocytes (EA) by peritoneal macrophages of (NZB X NZW)F1 or B/W mice is not intrinsic, but is related to the development of the autoimmune disease process. The defect appears to be confined to peritoneal macrophages, since bone marrow (BM)-derived macrophages have normal to elevated activities in vitro. The peritoneal macrophage defect is not due to blockade of Fc receptors in vivo, as shown by long-term culture or recovery of phagocytic and binding activities after removal of Fc receptors by pronase, but represents a reduced number of receptors with slightly delayed turnover. The defect can be reversed by elicitation of activated macrophages with Corynebacterium parvum, thioglycollate, or proteose peptone in vivo. Normal Fc-mediated phagocytosis and binding by BM-derived macrophages cultured from untreated autoimmune mice is enhanced by pretreatment of mice with C. parvum, thioglycollate, or proteose peptone. The cause of the defect in Fc-mediated phagocytosis by resident peritoneal macrophages of autoimmune mice was not ascertained; it may be due to abnormal macrophage kinetics or to the local effects of lymphokines released as a result of other autoimmune changes.     

Streptococcus suis interactions with the murine macrophage cell line J774: adhesion and cytotoxicity

Streptococcus suis capsular type 2 is an important etiological agent of swine meningitis, and it is also a zoonotic agent. Since one hypothesis of the pathogenesis of S. suis infection is that bacteria enter the bloodstream and invade the meninges and other tissues in close association with mononuclear phagocytes, the objective of the present study was to evaluate the capacity of S. suis type 2 to adhere to macrophages. An enzyme-linked immunosorbent assay technique was standardized to simply and accurately measure the rate of bacterial attachment to phagocytic cells. Results were confirmed by plate counting. Adhesion was dependent on bacterial concentration and incubation time and was not affected by cytochalasin pretreatment of macrophages. Inhibition studies showed that the sialic acid moiety of the S. suis capsule would be, at least in part, responsible for bacterial recognition by macrophages. Serum preopsonization of bacteria increased adhesion levels. Complement would be partially implicated in the serum-enhanced binding of S. suis to cells. Adhesion varied among different S. suis type 2 isolates. However, high bacterial concentrations of several isolates were cytotoxic for cells, and these cytotoxic effects correlated with suilysin production. Indeed, hemolytic strain supernatants, as well as purified suilysin, reproduced cytotoxic effects observed with live bacteria, and these effects were inhibited by cholesterol pretreatment. Bacterial adhesion and cytotoxicity were confirmed by scanning and transmission electron microscopy. We hypothesize that attachment of bacteria to phagocytes could play an important role in the pathogenesis of S. suis infection by allowing bacterial dissemination and causing a bacteremia and/or septicemia. This interaction could also be related to the activation of the host inflammatory response observed during meningitis.     

Effect of phagocytosis of erythrocytes and erythrocyte ghosts on macrophage phagocytic function and hydrogen peroxide production

Our previous studies have shown that an in vivo phagocytic challenge with IgG-coated erythrocytes can depress Kupffer cell complement and Fc receptor function, as well as decrease the survival rate following endotoxemia and bacteremia. In an effort to better understand the mechanism underlying these in vivo findings, the present study evaluated the in vitro effects of a phagocytic challenge with either IgG-coated erythrocytes (EIgG) or erythrocyte ghosts (GIgG) on macrophage phagocytic and respiratory burst activity. Elicited rat peritoneal macrophage (PM) monolayers were challenged with varying doses of EIgG, then the noninternalized EIgG were lysed hypotonically and the monolayers incubated for an additional hour prior to determining phagocytic function and PMA-stimulated hydrogen peroxide production. Challenge of PM with 1×10⁶ EIgG per well had no effect, but challenge with 1×10⁷ or 1×10⁸ EIgG per well caused a dose-dependent depression of phagocytic function or hydrogen peroxide production. GIgG were formed by hypotonically lysing EIgG bound to PM at 4°C. The bound GIgG were phagocytized during a subsequent incubation at 37°C. Challenge with GIgG depressed phagocytic function only with the highest challenge dose tested (1×10⁸ per well) and did not depress hydrogen peroxide production. The observation that prior phagocytic challenge with EIgG depressed macrophage function to a greater extent than challenge with GIgG supports our previous in vivo observations. Furthermore, these studies suggest that the internalization of erythrocyte contents, and not phagocytosis per se, plays an important role in determining macrophage host defense function.     

2.4G2, a monoclonal antibody to macrophage Fc gamma receptors, reacts with murine T cell Fc gamma receptors and IgG-binding factors

In order to determine whether T cell Fc gamma receptors (Fc gamma R) and IgG-binding factors (IgG-BF) are structurally related, we searched for common antigens on these molecules. We found that the anti-macrophage Fc gamma 1/gamma 2bR monoclonal antibody 2.4G2 binds to similar determinant(s) on the macrophage-like J774 cells and on the Fc gamma R+ hybridoma T cells T2D4. On the T cell membrane, these determinants are associated with Fc gamma 1/gamma 2bR. They are absent on Fc gamma R- hybridoma T cells or the FcR- BW5147 thymoma cells. 2.4G2-reactive molecules were also detected in soluble material either extracted or released in the supernatant of Fc gamma R+ hybridoma T cells. 2.4G2-reactive molecules released in the supernatant of T2D4 cells could be absorbed on Sepharose beads coupled to rabbit IgG and they were recovered by acid elution. Conversely, Sepharose beads coupled to 2.4G2 retained molecules which had affinity for rabbit IgG, which suppressed an in vitro antibody response and which had the same molecular weight as IgG-BF. These results indicate that T cell Fc gamma R and IgG-BF share common epitopes and that 2.4G2 can serve as an anti-IgG-BF antibody.