Dietary conjugated linoleic acid decreased cachexia,macrophage tumor necrosis factor-alpha production,and modifies splenocyte cytokines production

The effect of conjugated linoleic acid (CLA) on macrophage functions were studied in vitro, in vivo, and ex vivo. In RAW macrophage cell line, CLA (mixed isomers) was shown to inhibit lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) production. Two CLA isomers, c9,t11 and t10,c12, were tested on RAW cells and it was found that the c9,t11 was the isomer responsible for the inhibition of LPS-induced TNF-alpha production. BALB/c mice were used to determine the effect of dietary CLA on body weight wasting and feed intake after LPS injection. CLA was protective against LPS-induced body weight wasting and anorexia. Plasma TNF-alpha levels after LPS injection were lower in the CLA group compared with the corn oil-fed control group 2 hr post-LPS injection. In a separate experiment, 30 mice were fed a CLA-supplemented diet or a corn oil-supplemented diet for 6 weeks and peritoneal resident macrophages were obtained for measuring TNF-alpha and nitric oxide production after in vitro exposure to interferon-gamma (IFN-gamma) and/or LPS. TNF-alpha production was not found to be different in peritoneal macrophages from mice fed the dietary treatments, but less nitric oxide was produced in macrophages from CLA-fed mice upon stimulation when compared with macrophages from control-fed mice. Splenocytes were also collected from the mice fed the dietary treatments and stimulated to produce cytokines in culture. Supernatant was used to run cytokine enzyme-linked immunoabsorbant assays. Interleukin-4 (IL-4) was decreased in CLA-fed mice when splenocytes were stimulated with concanavalin A (Con A) for 44 hr; however, IL-2 and the IL-2-to-IL-4 ratio were elevated.     

Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.     

Role of bacterial DNA in macrophage activation by group B streptococci

Bacterial DNA (CpG DNA) induces macrophage activation and the production of inflammatory mediators, including tumor necrosis factor (TNF) and nitric oxide (NO) by these cells. However, the role of bacterial DNA in the macrophage response to whole bacteria is unknown. We used overlapping strategies to estimate the relative contribution of bacterial DNA to the upregulation of TNF and NO production in macrophages stimulated with antibiotic-treated group B streptococci (GBS). Selective inhibitors of the bacterial DNA/TLR9 pathway (chloroquine, an inhibitory oligonucleotide, and DNase I) consistently inhibited GBS-induced TNF secretion by 35-50% in RAW 264.7 macrophages and murine splenic macrophages, but had no effect on inducible nitric oxide synthase (iNOS) accumulation or NO secretion. Similarly, splenic and peritoneal macrophages from mice lacking TLR9 expression secreted 40% less TNF than macrophages from control mice after GBS challenge but accumulated comparable amounts of iNOS protein. Finally, studies in both RAW 264.7 cells and macrophages from TLR9-/- mice implicated GBS DNA in the upregulation of interleukins 6 (IL-6) and 12 (IL-12) but not interferon-beta (IFNbeta), a key intermediary in macrophage production of iNOS/NO. Our data suggest that the bacterial DNA/TLR9 pathway plays an important role in stimulating TNF rather than NO production in macrophages exposed to antibiotic-treated GBS, and that TLR9-independent upregulation of IFNbeta production by whole GBS may account for this difference.     

Effect of cytokines and growth factors on the macrophage colony-stimulating factor secretion by human bone marrow stromal cells

We have investigated the effect of growth factors, inflammatory and anti-inflammatory cytokines on the macrophage colony-stimulating factor (M-CSF) secretion by cultured human bone marrow stromal cells. Their production of M-CSF cultured in serum-free medium is enhanced in a time-dependent manner in response to tumour necrosis factor (TNF-)alpha and interleukin (IL-)4 but not to IL-1, IL-3, IL-6, IL-7, IL-10, SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, bFGF and transforming growth factor (TGF-)beta. The co-addition of IL-4 and TNF-alpha has a greater than additive effect on the secretion of M-CSF suggesting that they act synergistically. The anti-inflammatory molecules IL-10 and TGF-beta have no effect on the TNF-alpha-induced M-CSF synthesis by marrow stromal cells. In conclusion TNF-alpha and IL-4 are potent stimulators of the M-CSF synthesis by human bone marrow stromal cells, a result of importance regarding the role of M-CSF in the proliferation/differentiation of mononuclear-phagocytic cells and the role of marrow stromal cells as regulators of marrow haematopoiesis.     

Keratinocyte-derived granulocyte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages

Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.     

Activation of adenosine A(3) receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells

Adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been tested from the point of view of potentiating the effects of hematopoietic growth factors interleukin-3 (IL-3), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) on the growth of hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in suspension of normal mouse bone marrow cells in vitro. IB-MECA alone induced no GM-CFC growth. Significant elevation of numbers of GM-CFC evoked by the combinations of IB-MECA with IL-3, SCF, or GM-CSF as compared with these growth factors alone has been noted. Combination of IB-MECA with G-CSF did not induce significantly higher numbers of GM-CFC in comparison with G-CSF alone. Joint action of three drugs, namely of IB-MECA + IL-3 + GM-CSF, produced significantly higher numbers of GM-CFC in comparison with the combinations of IB-MECA + IL-3, IB-MECA + GM-CSF, or IL-3 + GM-CSF. These results give evidence of a significant role of selective activation of adenosine A(3) receptors in stimulation of the growth of granulocyte/ macrophage hematopoietic progenitor cells.     

TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages

The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO-), leading to macrophage apoptosis. Since TNF-alpha and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-alpha and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1-/- peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-alpha+Nramp1+/+ and B10R, and more IL-10+Nramp1-/- and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-alpha inhibited apoptosis, diminished NO- production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO- production. Murine rTNF-alpha induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO- production and caspase 1 activation. NO- and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO- production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-alpha- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-alpha-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival.     

Stimulation, inhibition and death of macrophages infected with Trichophyton rubrum

Trichophyton rubrum is the most common pathogen causing dermatophytosis, accounting for approximately 80% of the reported cases of onychomycosis. Since 90% of the chronic dermatophyte infections are caused by T. rubrum, it is likely that this pathogen must have evolved mechanisms that evade or suppress cell-mediated immunity. Several reports have highlighted the participation of phagocytes in the immune defense against fungi; however, few studies have addressed the role of these cells in dermatophytosis. In this study, we investigated the interactions of resident and peritoneal macrophages with T. rubrum. We show here that the interaction of T. rubrum conidia with resident macrophages results in the production of TNF-alpha and IL-10 but not IL-12 and nitric oxide. Infected macrophages down-regulated the expression of co-stimulatory molecules (CD80 and CD54). We also show that phagocytosis of T. rubrum conidia is inhibited by the addition of fungal exoantigens or mannan. Cytotoxicity assays indicated that after 8 h of conidia ingestion macrophage viability decreased drastically. Electron microscopy revealed that the ingested conidia grow and differentiate into hyphae inside macrophages leading to rupture of the macrophage membrane.     

Lactoferrin feeding augments peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans

Oral administration of lactoferrin (LF), an innate-defense protein present in exocrine secretions such as milk and in neutrophils, is reported to improve host-protection against infections with microorganisms including pathogenic fungi, possibly due to an immunomodulatory effect. This study aimed to evaluate the effect of bovine LF feeding on peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans. Time course analysis during the 14 days following Candida-priming revealed that LF administration slightly increased the number of peritoneal exudate cells, and significantly enhanced the production of superoxide anion (O2(-)) and nitric oxide (NO) by peritoneal macrophages at day 7. LF administration facilitated NO production and Candida hyphal-growth inhibition by macrophages derived from Candida-primed mice but not non-primed mice, suggesting that the action of LF is dependent on the immune status of the host. LF administration altered the kinetics of cytokines in the peritoneal lavage fluid of Candida-primed mice. Enhancement of cytokine levels by LF was observed for IL-12 at day 5 and IFN-gamma at day 9, but not for TNF-alpha or IL-10. In conclusion, LF feeding augmented the activities of macrophages in a manner dependent on Candida-priming and these effects may be related to enhanced cytokine levels.     

Regulation of activated macrophage antimicrobial activities. Identification of lymphokines that cooperate with IFN-gamma for induction of resistance to infection

Macrophages exposed to lymphokines (LK) before exposure to parasites develop the capacity to resist infection with amastigotes of Leishmania major. Activity of LK for induction of this activated macrophage effector function is abrogated by depleting the LK of IFN-gamma, yet IFN-gamma is incapable of inducing the activity by itself. To identify the factors in LK that serve as second signals for induction of resistance to infection, we exposed macrophages to the following cytokines available as recombinant or highly purified reagents: CSF-1, granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-1, -2, -3, -4, and -5, and IFN-alpha/beta. None of these factors induced resistance to infection by themselves or in combination with each other; in the presence of 50 U/ml IFN-gamma, three cytokines were active: GM-CSF, IL-2, and IL-4. IFN-gamma was an essential component of the activation cascade but was insufficient by itself to induce the effector reaction. Cytokines that act as cofactors with IFN-gamma worked directly on macrophages and not through another cell in the peritoneal cell (PC) cultures. Activation of PC depleted of Thy-1.2+ cells (85 +/- 5% macrophages) and bone marrow-derived macrophages (100% macrophages) showed that 50% maximal doses of GM-CSF, IL-2, and IL-4 for these macrophage-enriched populations were not different than for untreated PC. Unlike other effector reactions of activated macrophages, bacterial LPS did not synergistically enhance the activity of any of the cytokines, alone or in combination with IFN-gamma. Antibody depletion of the active cytokines from LK, singly or in combination, failed to alter the dose response of the active factors in whole LK for induction of resistance to infection. Thus, multiple factors can provide the second signal for IFN-gamma in the induction of resistance to infection, namely, GM-CSF, IL-2, IL-4, and at least two additional undefined factors in whole LK. Resistance to infection may be the first example of an activated macrophage effector reaction that has an absolute requirement for more than one endogenous signal for its induction.     

Response of resident murine peritoneal macrophages to in vivo administration of granulocyte-macrophage colony-stimulating factor

The effect of s.c. inoculation of purified recombinant derived granulocyte-macrophage (GM)-CSF on resident murine peritoneal macrophages was assessed in this study. From 18 to 24 h after s.c. administration of GM-CSF to normal mice, the resident peritoneal macrophages were harvested and the levels of membrane-bound IL-1, FcR, Mac-1 cell-surface Ag, and class II MHC expression were assessed. Peritoneal cells from GM-CSF-inoculated mice had significantly greater levels of membrane-bound IL-1 than did control mice. In addition when resident peritoneal macrophages from normal mice were purified by adherence and grown in the presence of GM-CSF, they produced greater levels of both membrane-bound and secreted IL-1. The peritoneal cells from GM-CSF-inoculated mice did not differ from controls in the expression of class II MHC-encoded Ag. This observation was confirmed by the finding that GM-CSF was unable to induce class II MHC expression on P388D1 cells, whereas a secondary mixed leukocyte culture supernatant was. Peritoneal cells from GM-CSF-inoculated mice also exhibited greater levels of expression of FcR and the Mac-1 cell-surface Ag. This resulted in an increase in their ability to phagocytose opsonized SRBC in vitro.     

Role of TNF-alpha in the induction of fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by IL-12 and IL-18

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.     

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.     

12/15-Lipoxygenase regulates the inflammatory response to bacterial products in vivo

The peritoneal macrophage (Mphi) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11b(high) cells, with the remaining 5% being 12/15-LOX(-). 12/15-LOX(+) cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX(-) cells are a dendritic cell population. Resident peritoneal Mphi numbers were significantly increased in 12/15-LOX(-/-) mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mphi from 12/15-LOX(-/-) mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX(-/-) mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX(-/-) adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX(+) cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX(-/-) mice showed elevated TGF-beta1, along with increased immigration of monocytes/Mphi, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-alpha. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MPhi population and regulates both the recruitment of monocytes/Mphi and cytokine response to bacterial products in vivo.     

Dectin-1 expression and function are enhanced on alternatively activated and GM-CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and lipopolysaccharide

Dectin-1 is the major macrophage receptor for beta-glucans and generates a proinflammatory response through the recognition of these carbohydrates on fungal pathogens. We have examined the effects of cytokines and other agents on the expression and functions of dectin-1 in both resident and elicited murine peritoneal macrophages (Mphi). Dectin-1 expression was found to be highly up-regulated by GM-CSF and by the cytokines that induce alternative macrophage activation, IL-4 and IL-13. In contrast, IL-10, LPS, and dexamethasone, but not IFN-gamma, down-regulated the expression of this receptor. Modulation of dectin-1 receptor levels correlated with the ability of these macrophages to bind zymosan and significantly affected the contribution of this receptor to the resultant proinflammatory response, as measured by the production of TNF-alpha, although some Mphi-specific differences were observed. These results correlate with the known effects of these cytokines and other agents on the ability of the immune system to recognize and respond to fungal pathogens.