Phorbol ester-stimulated superoxide production by murine bone marrow-derived macrophages requires preexposure to cytokines

Murine resident peritoneal macrophages (RPM) generate superoxide (O2-) in response to stimulation with PMA or zymosan. Murine bone marrow-derived macrophages (BMM) generate O2- in response to zymosan but not PMA. However, the ability to generate O2- in response to PMA could be induced in BMM by pre-exposing the cells to certain cytokines, including granulocyte-macrophage CSF (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, and, to a lesser extent, IL-1 alpha. Bacterial LPS also induced the ability to respond to PMA. These same agents were also shown to prime RPM for enhanced PMA-induced respiratory burst. In contrast to GM-CSF, CSF-1 did not enhance the ability of BMM or RPM to generate O2- in response to PMA. Pretreatment with GM-CSF or TNF-alpha did not significantly affect the zymosan-induced release of O2- by BMM. These results suggest that unprimed BMM have a deficiency in the PMA-dependent signaling pathway that is corrected by exposure to selected cytokines. The results also raise the possibility that the basal ability of tissue macrophages to generate a respiratory burst in response to PMA may be a reflection of in vivo exposure to cytokines.     

Relationship between production and release of lymphocyte-activating factor (interleukin 1) by murine macrophages. 1. Effects of various agents

The relationship between the production and release of lymphocyte-activating factor (LAF, or interleukin 1) by cultured murine peritoneal macrophages (Mφ) was investigated. Unstimulated Mφ produce high levels of intracellular LAF within a few hours after culturing, but release little of this activity into their culture medium. Addition of various agents was found to increase significantly the production and release of LAF, with three different patterns: (a) Both intracellular and extracellular LAF activities were increased in response to latex beads, (b) A marked increase of intracellular LAF, with just a minimal elevation of extracellular activity, was stimulated by LPS. (c) Sharp increases in LAF release, with small increments of the intracellular activity, were induced by silica and glucocerebroside (GL₁). Silica and GL₁ damaged the cultured Mφ, as indicated by the increased release of lactate dehydrogenase. It is significant, therefore, that silica and GL₁ increased both intracellular and extracellular LAF levels, suggesting that damage of Mφ may stimulate total LAF production. A combination of LPS with silica or GL₁ acted synergistically on Mφ to release very high levels of LAF, which far exceeded those released by the individual agents. The agents were also tested on Mφ which were precultured, to deplete their LAF content. Latex, LPS, or silica increased LAF production and release by precultured Mφ, but the levels were lower than those obtained with freshly cultured Mφ. The results of this study thus show that the level of LAF release does not necessarily reflect the level of total LAF production by cultured Mφ and suggest that injurious agents may promote LAF production.     

Down-regulation of interleukin 1 production by macrophages of sarcoma-bearing mice

Peritoneal macrophages from mice bearing a transplantable methylcholanthrene-induced sarcoma produced progressively less IL 1 as tumor burden increased. The loss of activity was not explained by the production of any inhibitor of the mouse thymocyte comitogen bioassay. Immune precipitation with a polyclonal antibody confirmed the decline in IL 1 appearance. Although tumor-bearing animals lost approximately 17% of their carcass mass, the reduced production of IL 1 was not satisfactorily explained by coexistent malnutrition, since similarly depleted non-tumor-bearing mice were capable of producing IL 1. In addition to an altered IL 1 production by macrophages of tumor-bearing mice, SDS-polyacrylamide gel electrophoresis and autoradiography revealed that the pattern of secretory protein synthesis from LPS-stimulated and unstimulated peritoneal macrophages differed between tumor-bearing and control animals. Administration of LPS to tumor-bearing mice early after tumor transplantation resulted in reduced tumor growth and prevented the down-regulation of in vitro IL 1 production by peritoneal macrophages. These findings demonstrate a specific defect in IL 1 production associated with increasing tumor burden. Further studies are required to determine whether this defect in IL 1 synthesis contributes to the increased tumor growth.     

IFN-alpha beta-dependent, IFN-gamma secretion by bone marrow-derived macrophages controls an intracellular bacterial infection.

Several reports have indicated that cell lineages apart from NK and T cells can also express IFN-gamma. However, the biological relevance of this finding is uncertain. We show in this study that bone marrow-derived macrophages (BMMs) express IFN-gamma at the mRNA and protein level early after infection with Chlamydia pneumoniae. Increased IFN-gamma mRNA accumulation by infected BMMs is early, transient, and requires both bacterial and host protein synthesis. The induction of IFN-gamma mRNA levels is independent of IL-12 and was dramatically enhanced in IL-10(-/-) BMMs. Such IL-10(-/-) BMMs contained less bacteria than the wild-type controls, whereas IFN-gammaR(-/-) BMMs showed increased C. pneumoniae load. Inducible NO synthase (iNOS) also participates in the control of bacterial load, as shown by the enhanced numbers of C. pneumoniae in iNOS(-/-) BMMs. However, the increased accumulation of iNOS mRNA and NO in C. pneumoniae-infected BMMs depended on the presence of IFN-alphabeta, but was independent of IFN-gamma. Interestingly, IFN-alphabeta are also required for increased IFN-gamma mRNA accumulation in C. pneumoniae-infected BMMs. Accordingly, IFN-alphabetaR(-/-) BMMs showed higher levels of C. pneumoniae than wild-type BMMs. Our findings unravel an autocrine/paracrine macrophage activation pathway by showing an IFN-alphabeta-dependent IFN-gamma and iNOS induction in response to infection, which protects macrophages against intracellular bacterial growth.     

Leukotrienes do not regulate interleukin 1 production by activated macrophages

The objective of this work was to investigate the role of leukotrienes in the production of IL-1 by activated human peripheral blood monocytes and mouse peritoneal macrophages. Using overnight adherent macrophages, stimulation with lipopolysaccharide or zymosan caused a time-dependent increase in IL-1 production. LTC4 was detected and preceded IL-1 production only in zymosan-treated macrophages. Lipopolysaccharide did not stimulate macrophages to produce LTC4. Zymosan-stimulated LTC4 production was inhibited by the lipoxygenase inhibitors, ICI207968 (3.20 microM), nordihydroguaiaretic acid (0.22 microM), phenidone (4.60 microM), REV5901 (0.20 microM), and the Merck 5-lipoxygenase "translocation inhibitor" MK886 (0.02 microM) with IC50 values as shown in parenthesis. However, none of these inhibitors reduced IL-1 production at concentrations which completely inhibited leukotriene synthesis. Taken together, these results do not support a role for leukotrienes in the production of IL-1 by zymosan-activated macrophages.     

Genetic regulation of lipopolysaccharide-induced interleukin 1 production by murine peritoneal macrophages

The production of IL 1 by LPS-stimulated peritoneal macrophages from inbred mouse strains was studied. Macrophages from A/J (A) mice were deficient in IL 1 production, when compared with high IL 1-producing strains, including C57BL/6J (B). The difference between A and B macrophages was maintained over a wide LPS concentration range and throughout a 72-hr incubation period. Because of these differences, it was possible to investigate the mechanisms regulating IL 1 production by applying techniques of genetic analysis by using recombinant inbred (RI) strains derived from the A and B progenitors. A strain distribution pattern (SDP) of IL 1 production (low/high response) was obtained with the use of 15 AXB/BXA RI strains. This suggested the presence of a major gene locus controlling the production of IL 1 in response to LPS stimulation, with allelic differences presumably resulting in deficient or efficient IL 1 production. In addition, there appeared to be one or more other loci involved in determining the magnitude of the IL 1 response to LPS in the responder mice. The IL 1 response did not appear to be linked to the major histocompatibility complex, since B10.A mice (which share the same H-2a haplotype as A/J) were efficient IL 1 producers. There did not appear to be any correlation between the degree of IL 1 production and the magnitude of the peritoneal macrophage inflammatory response, or between IL 1 production and LPS responsiveness (as determined by splenocyte proliferation). SDP analysis also indicated that the IL 1 response was not linked to macrophage tumoricidal activity. A comparison of the SDP for IL 1 production with a library of SDP for other known genetic waits suggested linkage with at least four loci on chromosome 1.     

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.     

Induction by lipopolysaccharide of intracellular and extracellular interleukin 1 production: analysis with synthetic models

An attempt was made to identify the molecular structures that are present in bacterial LPS and induce the production of intracellular and extracellular pools of IL 1 by peritoneal macrophages of the mouse and by human monocytes. Activities of glycolipids and carbohydrates prepared by synthesis, and structurally related to the hydrophobic (Lipid A) and to the polysaccharide (PS) regions of LPS were compared with those induced by Bordetella pertussis endotoxin and by fragments derived therefrom. Both isolated regions of this LPS (PS and Lipid A) were able to induce IL 1 synthesis by monocytes and macrophages. Among the synthetic glycolipids employed, propyl-2-deoxy-2-[(3R)-3-hydroxytetrade-canamido]-4-O-pho sph ono-6-O-tetradecanoyl-beta-D-glucopyranoside (glycolipid M9) induced IL 1 secretion more efficiently than Lipid A and LPS, whereas the amounts of intracellular IL 1 produced upon induction by these three substances were comparable. Macrophages from C3H/HeJ mice were unresponsive to Lipid A and to glycolipid M9, but produced IL 1 when incubated with PS or with a hydrophilic fragment isolated after methanolysis of the endotoxin. However, all synthetic derivatives of 3-deoxy-D-manno-2-octulosonic acid (KDO) used in this study failed to induce IL 1 production by both mouse macrophages and human monocytes. The implications of these findings for a more precise comprehension of the molecular mechanism of LPS-induced activation of macrophages, and the relations between the molecular structures required for the induction of IL 1 production vs cytostatic activity in macrophages, are discussed.     

Cytokine production by M-CSF- and GM-CSF-induced mouse bone marrow-derived macrophages upon coculturing with late apoptotic cells

Recently, we found that resident peritoneal macrophages produce MIP-2, one of the major chemokines for neutrophils, upon coculturing with late apoptotic cells, and that intraperitoneal injection of late apoptotic cells into the peritoneal cavity causes neutrophil infiltration via MIP-2. It is not known, however, whether or not macrophages are heterogeneous in such MIP-2 production. In this study, we examined changes in the surface phenotype during the differentiation of bone marrow cells into macrophages due to M-CSF and GM-CSF, and then examined the production of cytokines, namely IL-12 p40, MIP-2, IL-10, and TGF-β, following phagocytosis of late apoptotic cells with these macrophages or LPS stimulation of these macrophages. GM-CSF and M-CSF induced macrophage populations with distinct but overlapping cell surface phenotype. Although these macrophages phagocytosed late apoptotic cells to a similar extent, they produced either IL-12 p40 or IL-10, whereas they produced MIP-2 to a similar extent after the coculture, raising the possibility that late apoptotic cells may induce neutrophil infiltration in any organs, such as the liver and lungs, where the macrophages are differentiated by either M-CSF or GM-CSF, respectively.     

CSF-1 stimulates glucose uptake in murine bone marrow-derived macrophages

3H-2-deoxyglucose was used as an isotopic tracer for the measurement of glucose uptake into quiescent murine bone marrow derived macrophages. A purified colony stimulating factor (CSF-1) was shown to stimulate 3H-2-deoxyglucose uptake in a dose-dependent manner. This stimulation was rapid, with a maximal effect seen at 20-30 minutes after growth factor addition. Both the inhibition by cytochalasin B and also the relative degree of competition by high concentrations of a series of glucose analogues suggest that the basal and CSF-1 stimulated 2-deoxyglucose uptake occur via a carrier facilitated D-glucose transport system. The data indicate that a purified growth factor can increase the glucose uptake in macrophages, a finding which could be relevant to the survival and/or the proliferative response of this and other haemopoietic cell types.     

The interaction of 125I-colony-stimulating factor-1 with bone marrow-derived macrophages

The colony-stimulating factor, CSF-1, stimulates cultured quiescent murine bone marrow-derived macrophages (BMM) to enter DNA synthesis with a lag phase of 10-12 h. The binding, dissociation, internalization, and degradation of 125I-CSF-1 by BMM during the lag phase were investigated. Quiescent BMM express approximately 5 X 10(4) cell surface receptor sites/cell but contain additional cryptic sites (approximately 10(5)/cell) that can appear at the cell surface within 10 min at 37 degrees C. Studies of the binding reaction at both 2 degrees C (Kd less than or equal to 2 X 10(-13) M) and 37 degrees C (Kd approximately 4 X 10(-10) M) are consistent with the existence of a single class of cell surface sites. The disappearance of cell surface 125I-CSF-1 following a 2-37 degrees C temperature shift results from two, competitive, first order processes, internalization and dissociation. Internalization (t1/2 = 1.6 min) is 6 times more frequent than dissociation (t1/2 = 9.6 min). Following internalization, 10-15% of the intracellular CSF-1 is rapidly degraded whereas the remaining 85-90% is slowly degraded by a chloroquin-sensitive first order process (t1/2 greater than 3.5 h). These findings were confirmed and extended by studies of the uptake of 125I-CSF-1 at 37 degrees C. Following addition of 125I-CSF-1, cell surface receptors are rapidly down-regulated (t1/2 approximately 7 min) and their replacement does not commence until 20-60% of pre-existing surface receptor sites have disappeared. Despite receptor replacement, initially from the cryptic pool and later by de novo synthesis and/or receptor recycling (4 molecules/cell/s at steady state), the number of receptors at the cell surface remains low. The process results in the intracellular accumulation of large amounts of 125I-CSF-1 (greater than 10(5) molecules/cell) by BMM. Thus, whereas the kinetics of association, dissociation, and internalization of CSF-1 with BMM and peritoneal exudate macrophages are similar, BMM, which exhibit a higher proliferative response, degrade growth factor 12 times more slowly.     

The intestinal microflora regulates cytokine production positively in spleen-derived macrophages but negatively in bone marrow-derived macrophages.

Besides its role as a barrier against potential pathogens, intestinal flora is presumed to protect the host by priming the immunological defense mechanisms. In this respect, the influence of intestinal flora on macrophage precursors was examined, and its modulating effect was compared on LPS-induced cytokine production by macrophages derived from bone marrow and spleen precursors (BMDM and SDM respectively). The regulation of IL-1, IL-6, TNF-alpha and IL-12 production in macrophages from germ-free and from three groups of flora-associated mice, conventional, conventionalized and E. coli-mono-associated mice, was investigated. The whole flora inhibited IL-1, TNF-alpha and IL-12 secretion by BMDM, whereas it had a stimulatory effect on IL-12 secretion by SDM. Implantation of E. coli alone enhanced cytokine secretion by BMDM but had a more limited effect than whole flora on SDM, enhancing only TNF-alpha and IL-12 secretion. Study of expression of mRNA showed a correlation with protein secretion for IL-6 but not for TNF-alpha and IL-1. IL-12 enhancement in BMDM seemed to be dependent on regulation of p35 mRNA expression while it was correlated to increased p40 mRNA expression in SDM. The results demonstrated that intestinal flora modulated bone marrow and spleen macrophage cytokine production in a differential manner and suggested a role for bacteria other than E. coli among the whole flora. The contrasting effects exerted by the intestinal flora on bone marrow and spleen precursors are an interesting observation in view of the different functions of these organs in immunity. The finding that intestinal flora enhanced IL-12 production in spleen is also potentially important since this cytokine is implicated in the determination of the relative levels of Th1 and Th2 responses and plays a pivotal role in host defense against intracellular microorganisms.     

Control of arachidonic acid accumulation in bone marrow-derived macrophages by acyltransferases

The turnover of phospholipid fatty acid moieties of bone marrow-derived macrophages was analyzed by separate determination of degrading and acylating activities. Acylating activities were followed in intact cells by incubation with excess arachidonic acid and degradation of phospholipids was followed in cells prelabeled with fatty acids. Significant phospholipase A2 activity was detectable only if the reutilization of liberated fatty acid was inhibited , e.g. by p-chloromercuribenzoate. It was of interest that the divalent cation ionophore A 23187 and various antiphlogistic drugs like indomethacin, diclofenac, and acetylsalicylic acid were found to inhibit the acylation reaction. These compounds led to increased levels of free arachidonic acid in stimulated, as well as in unstimulated cells. Increased activities of phospholipase A2 were achieved by treatment with the bivalent cation ionophore A 23187 and with zymosan. The effect of zymosan obtained from various sources was found to be exclusively due to contamination of tee zymosan particles with phospholipase A2 activity. Even when the cellular phospholipase activity was increased by the addition of exogenous phospholipase activity contained in the zymosan particles, degradation of cellular phospholipids was not measurable unless the reacylation was inhibited. These results suggest that in the cells studied, the level of free arachidonic acid is mainly controlled by the activity of the lysophosphatide acyltransferase.     

Biodegradable chitosan particles induce chemokine release and negligible arginase-1 activity compared to IL-4 in murine bone marrow-derived macrophages

Alternatively activated macrophages have been implicated in the therapeutic activity of biodegradable chitosan on wound healing, however, the mechanisms of phenotypic differentiation are still unclear.In vitro, macrophages stimulated with high doses of chitosan (?500 μg/mL) were reported to produce low-level markers associated with alternative activation (arginase-1) as well as classical activation (nitric oxide), and to undergo apoptosis. In this study, we tested the hypothesis that 40 kDa biodegradable chitosan (5–500 μg/mL) is sufficient to polarize mouse bone marrow-derived macrophages (BMDM) in vitro to an alternatively activated phenotype. Control cultures were stimulated with IL-4 (alternative activation), IFN-γ/LPS (classical activation), 1 μm diameter latex beads (phagocytosis), or left untreated. After 48 h of in vitro exposure, BMDM phagocytosed fluorescent chitosan particles or latex beads, and remained viable and metabolically active, although some cells detached with increasing chitosan and latex bead dosage. Arginase-1 was over 100-fold more strongly induced by IL-4 than by chitosan, which induced only sporadic and weak arginase-1 activity over untreated BMDM, and no nitric oxide. IFN-γ/LPS stimulated nitric oxide production and arginase-1 activity and high concentrations of inflammatory cytokines (IL-6, IL-1β, TNF-α, MIP-1α/MIP-1β), while latex beads stimulated nitric oxide and not arginase-1 activity. Chitosan or latex bead exposure, but not IL-4, tended to promote the release of several chemokines (MIP-1α/β, GM-CSF, RANTES, IL-1β), while all treatments promoted MCP-1 release. These data show that chitosan phagocytosis is not sufficient to polarize BMDM to the alternative or the classical pathway, suggesting that biodegradable chitosan elicits alternatively activated macrophages in vivo through indirect mechanisms.     

Synthesis of interleukin 1beta and interleukin 6 by stimulated rat peritoneal macrophages: modulation by glutamine

Synthesis and secretion of IL-1beta and IL-6 were compared in LPS-stimulated rat peritoneal macrophages, and the effect of glutamine studied. LPS induced a parallel increase in mRNA and synthesis of IL-1beta and IL-6. IL-1beta accumulated mainly in the cytosol and IL-6 in the culture medium. Glutamine addition increased the synthesis of both cytokines, but the overall production (intra-+extracellular) of IL-1beta increased two-fold, although that of IL-6 increased only 1.3-fold. The influence of glutamine is discussed.     

Lipopolysaccharide-induced IL-1 beta production by human uterine macrophages up-regulates uterine epithelial cell expression of human beta-defensin 2

The uterine endometrium coordinates a wide spectrum of physiologic and immunologic functions, including endometrial receptivity and implantation as well as defense against sexually transmitted pathogens. Macrophages and epithelial cells cooperatively mediate innate host defense against bacterial invasion through the generation of immunologic effectors, including cytokines and antimicrobial peptides. In this study, we demonstrate that stimulation of peripheral blood monocytes and uterine macrophages with bacterial LPS induces the production of biologically active proinflammatory IL-1beta. High doses of estradiol enhance LPS-induced IL-1beta expression in an estrogen receptor-dependent manner. Furthermore, both peripheral blood monocyte- and uterine macrophage-derived IL-1beta induce secretion of antimicrobial human beta-defensin 2 by uterine epithelial cells. These data indicate dynamic immunologic interaction between uterine macrophages and epithelial cells and implicate a role for estradiol in the modulation of the immune response.     

Macrophage activation for the production of immunostimulatory cytokines by delivering interleukin 1 via biodegradable microspheres

Interleukin 1alpha (IL-1alpha), a pleiotropic cytokine with multiple anti-tumour activities, has been investigated in our laboratory for its potential to serve as an immunotherapeutic agent. In the present study, an attempt was made to direct IL-1alpha to macrophages, in order to induce their immunoregulatory activities. For that purpose, IL-1alpha was encapsulated within biodegradable poly(lactic/glycolic acid) microspheres, 1-5 microm diameter in size. The microspheres were efficiently taken-up by macrophages in culture and after intraperitoneal injection into mice. In culture, phagocytosis of the microspheres reached saturation within 3 h and there was no apparent effect of polymer type on the extent of uptake. In vivo uptake of human IL-1alpha-microspheres by the macrophages lead to cell activation, as evidenced by the enhanced production of murine IL-1alpha, IL-6 and IL-12. Control microspheres, containing bovine serum albumin, induced only background to low levels of cytokine production. These cytokines, when expressed by or secreted from macrophages, may stimulate in situ diverse immune and inflammatory responses, including T cell-mediated immune responses, such as the development of Th(1)cells and cytotoxic lymphocytes. Thus, directing IL-1alpha into macrophages, via the appropriate microspheres, may serve as a unique mean to activate these cells to participate in anti-tumour immune responses in situ.