MEKK3 is essential for lipopolysaccharide-induced interleukin-6 and granulocyte-macrophage colony-stimulating factor production in macrophages

Mitogen-activated protein/ERK kinase kinase 3 (MEKK3) is a Ser/Thr protein kinase belonging to the MEKK/STE11 subgroup of the MAP3K family. Recently, we found that MEKK3 plays a critical role in interleukin-1 (IL-1) receptor and Toll-like receptor 4 signalling using established primary mouse embryonic fibroblast (MEF) cell lines. However, the function of MEKK3 in immune cells has not been studied because germ-line MEKK3 knockout mice are embryonically lethal between embryonic days 10 and 11. In this study, we used small interference RNA to the mouse Mekk3 gene to specifically knock down MEKK3 expression in the macrophage line Raw264.7. We found that the lipopolysaccharide-induced IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production was dramatically decreased in MEKK3 knockdown cells whereas the tumour necrosis factor-alpha and IL-1beta production were not affected. We also observed that the ERK1/2, p38 and JNK MAPK induction in MEKK3 knockdown cells were moderately inhibited within the first 60 min of stimulation, while the ERK and p38 were more severely inhibited after 2-4 hr of stimulation. Degradation of IkappaBalpha was also partially blocked in MEKK3 knockdown cells. Notably, the impairment in IL-6 and GM-CSF production in the MEKK3 knockdown cells was restored by reintroducing a human Mekk3 cDNA that could not be targeted by mouse Mekk3-siRNAs. In conclusion, this study showed that MEKK3 is a crucial and specific regulator of the proinflammatory cytokines IL-6 and GM-CSF in macrophages and provided a novel method for investigating MEKK3 function in other immune cells.     

Role of macrophages in serum colony-stimulating factor induction by Lactobacillus casei in mice.

Heat-killed Lactobacillus casei YIT9018 (LC9018), when injected intravenously into mice at a dose of 4 to 40 mg/kg, induced the production of serum colony-stimulating factor (CSF). Since this induction was observed in both C3H/HeJ and C3H/HeN mice, LC9018 was considered to act differently from lipopolysaccharide. The amount of serum CSF induced by LC9018 in nude mice and whole-body-X-ray-irradiated mice was similar to that in control mice, but the induction of serum CSF was suppressed by the previous administration of carrageenan, indicating that macrophages, but not T cells, were responsible for serum CSF induction by LC9018. To determine whether macrophages themselves produce CSF or help other cells produce CSF in response to LC9018, we prepared adherent cells from the peritoneal cavity of normal mice and examined CSF activity in their conditioned media. Peritoneal adherent cells did not produce CSF without LC9018, but when cultivated with 1 mg of LC9018 per ml, they produced CSF at the same time that serum CSF was induced after the intravenous administration of LC9018. Additionally, in vitro-induced CSF formed macrophage, granulocyte, and mixed colonies, as serum CSF did. CSF production by peritoneal adherent cells was completely inhibited by cycloheximide (50 micrograms/ml), and neither the elimination of T cells from the peritoneal adherent cells by treating them with anti-Thy-1.2 antibody plus complement nor the addition of T cells affected CSF production. These results suggest that heat-killed LC9018 induces serum CSF in mice via direct stimulation of macrophages to produce CSF de novo.     

Spontaneous interaction in vitro between lymphocytes and syngeneic peritoneal macrophages of mice.

Ficoll-purified lymphocytes (peritoneal, splenic, or thymic) and macrophages (peritoneal) from Toxoplasma-immune and normal female NMRI mice were used. Suspensions of washed cells were made in medium 199 containing 20% heat-inactivated normal calf serum. Sixty minutes after the adherence of 10(5) macrophages to cover slips in Leighton tubes, lymphocytes were added in various concentrations. The mixed cellular population was then incubated at 37 C. Eighteen hours later, most of the lymphocytes were firmly attached to macrophages to form rosettes. This cellular interaction, which was temperature, cell ratio, and time dependent, occurred in the absence of any particular antigenic stimulation. Morever, the reaction was cytotoxic only for adhered lymphocytes as judged by staining with 0.2% trypan blue. Splenic and thymic lymphocytes were bound in significantly greater number than peritoneal lymphocytes. Incubation of macrophages for more than 48 h at 37 C before the addition of fresh lymphocytes markedly reduced rosette formation. Treatment of macrophages and lymphocytes with mouse anti-immunoglobulin did not affect the reaction. The labeling of lymphocytes with fluorescent anti-mouse sera and the use of nude NMRI mice showed that both B and T cells can form spontaneous rosettes with syngeneic peritoneal macrophages.     

Differential production of granulocyte-macrophage colony-stimulating factor by macrophages from mice susceptible and resistant to Leishmania mexicana amazonensis.

The present results demonstrate that macrophages from mice susceptible to infection with Leishmania mexicana amazonensis sustain a higher production of granulocyte-macrophage colony-stimulating factor (GM-CSF) throughout the in vitro infection than macrophages from a resistant strain. Resident macrophages from BALB/c and C57B1/10 mice were infected with promastigotes of L. mexicana amazonensis and the amount of biologically active GM-CSF was measured in the supernatants collected at different times of infection. Measurements were made by bone marrow and GM-CSF/interleukin-3 addicted cell proliferation. Because GM-CSF is a disease-exacerbating cytokine, its differential production by infected macrophages may be one of the mechanisms defining resistance or susceptibility to a leishmanial infection.     

Differences in lipopolysaccharide-induced signaling between conventional dendritic cells and macrophages

Dendritic cells (DCs) and macrophages contribute to the activation of immune responses against infectious agents. They sense the presence of microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs). Among the different PAMPs, the response to lipopolysaccharide (LPS) is one of the best characterized. Upon LPS encounter DCs undergo an activation process and acquire the ability to prime both natural killer and T-cell responses after migration to lymph nodes. Once they completed the effector phase, DCs reach a terminal differentiation stage and eventually die by apoptosis. By contrast, macrophages do not leave the tissue upon LPS recognition. They first initiate inflammatory processes and then switch to an anti-inflammatory phenotype to restore tissue homeostasis. In this review we will focus on the molecular bases of the divergent responses of DCs and macrophages to LPS.     

Interleukin-1beta induces in vivo tolerance to lipopolysaccharide in mice

Endotoxin or lipopolysaccharide (LPS) tolerance may be partially due to the secretion of potent anti-inflammatory cytokines following severe Gram-negative infections, or by low doses of LPS. In this work, we describe the effects of interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF-), two early cytokines secreted after LPS exposure, in the induction of LPS tolerance. Our results demonstrate that mice treated with three daily doses of 100 ng of IL-1 were tolerant to LPS-induced shock. However, TNF- was unable to induce an LPS refractory state. Given the fact that 100 ng of IL-1 increase the plasma levels of glucocorticoids, we evaluated whether a daily injection of dexamethasone (DEX) alone was able to reproduce the LPS-like tolerant state. However, no signs of LPS refractoriness were detected, except when DEX was administered concomitantly with a dose of IL-1 that does not induce corticosterone secretion (12 ng/mouse). This dose was found to induce in vitro up-regulation of the glucocorticoid receptors (GcR) of peritoneal macrophages following 24 h of treatment. In addition, we demonstrate that IL-1 is capable of inducing the down-regulation of Toll-like receptor 4 (TLR4), a crucial molecule in the signal transduction of LPS. Taken together, our results indicate that IL-1 can generate tolerance to LPS in vivo, and suggest that the regulation of mechanisms of the down-regulation of TLR4, as well as those involved in the expression of GcR and/or in the secretion of glucocorticoids, would be crucial for these effects.     

In vivo and in vitro effects of macrophage colony-stimulating factor (M-CSF) on bronchoalveolar macrophages for antihistoplasmal activity

The in vivo and in vitro effects of M-CSF on bronchoalveolar macrophages (BAM) activity against the intracellular fungal pathogen Histoplasma capsulatum (Hc) were studied. Three days after a single subcutaneous (s.c.) dose of M-CSF (2.5 mg/kg), enhanced ex vivo antifungal activity of BAM was measured. BAM from M-CSF-treated CD-1 mice significantly (P<0.01) inhibited the intracellular multiplication of He yeast cells in 20 h assays compared to BAM from control mice. This effect was not observed at days 1, 7, 11 or 21 post-treatment. A dose of 5 mg/kg s.c., but not 1 mg/kg, induced similar antifungal activity in BAM by day 3. Peritoneal macrophages (PM) from M-CSF-treated mice did not have enhanced antifungal activity at days and doses tested. BAM could also be activated for antihistoplasmal activity by M-CSF in vitro. M-CSF at 10,000 U/ml for 24 h or 5000 U/ml for 48 h induced significant (P<0.01) inhibition of intracellular multiplication of Hc. Interferon-gamma (IFN) plus lipopolysaccharide (LPS) activated BAM and PM in vitro to inhibit intracellular multiplication of He (P<0.001); the antihistoplasmal activity was completely inhibited by N^C-monomethyl l-arginine (N-MMA), indicating that an L-arginine-dependent nitric oxide-producing mechanism was operative. N-MMA could not inhibit the antihistoplasmal activity of BAM or PM activated by M-CSF in vitro. The mechanism by which M-CSF-activated macrophages inhibit intracellular multiplication of He remains to be determined.     

Granulocyte-macrophage colony-stimulating factor produced by splenic T lymphocytes of mice infected with Schistosoma japonicum.

The production of granulocyte-macrophage (GM) colony-stimulating factor (CSF) by splenic lymphocytes was examined in murine schistosomiasis japonica. When splenic lymphocytes obtained at various weeks after infection were cultured with soluble egg antigen, GM-CSF activity in the conditioned medium became detectable at 3 weeks after infection, reached a peak at week 5, and persisted at least up to week 7. Not only soluble egg antigen but also concanavalin A was highly effective in stimulating splenic lymphocytes to produce GM-CSF. When splenic lymphocytes were treated with anti-Thy-1.2 antibody and complement, GM-CSF-producing activity was completely abolished. The molecular weight of this T-cell-derived GM-CSF was estimated to be 30,000 by gel filtration on Sephadex G-150. After isoelectric focusing, GM-CSF activity was detected as two major peaks at pH 3.7 and 5.5. The physicochemical nature of this T-cell-derived GM-CSF was compared with those of known lymphokine GM-CSFs or with that of a previously reported GM-CSF in the serum of S. japonicum-infected mice (M. Owhashi and Y. Nawa, Infect. Immun. 49: 533-537, 1985).     

Platelet activating factor enhancement of lipopolysaccharide-induced tissue factor activity in monocytes: requirement of platelets and granulocytes.

Incubation of platelet activating factor (PAF) with heparinized blood caused no induction of tissue factor activity in monocytes. However, when PAF was added in addition to weak lipopolysaccharide (LPS), it amplified the LPS effect by 80%. By using separated fresh cell populations resuspended in plasma, PAF was shown to have no enhancing effect when added to mononuclear cells incubated with platelet-rich plasma in the presence of LPS. In contrast, when granulocytes also were included, PAF caused an almost 3-fold increase in LPS-induced tissue factor activity. A PAF antagonist blocked this effect and also reduced LPS-induced tissue factor activity of monocytes in whole blood in a dose-dependent manner. In the recombined cell incubation system, the maximal inhibition by the antagonist was observed in the presence of granulocytes. These data provide evidence for an effect of PAF on granulocytes that probably generates a product that, together with platelets, enhances LPS-induced tissue factor activity in monocytes.     

Inhibition of resistance to hemopoietic allo-grafts in granulocyte colony-stimulating factor transgenic mice

Transplanted allogeneic marrow cells often fail to engraft in a lethally irradiated host. This phenomenon, termed resistance to allogeneic marrow grafts or allo-resistance, is well documented, although its mechanism is not yet understood. Transplantation of major histocompatibility complex disparate allogeneic marrow cells into mice transgenic for granulocyte colony-stimulating factor (G-CSF) showed donor-derived spleen colonies (CFU-S) and resulted in stable allogeneic chimerism with excellent survival (100% up to 40 days and 89% up to 120 days). Under the same experimental conditions, all the littermate controls failed to show CFU-S and died shortly after marrow transplantation. Thus, resistance to allogeneic marrow cells appeared to be severely impaired in this transgenic mouse. The observation that neutralizing antibody against G-CSF restored allo-resistance in G-CSF transgenic mice and that CFU-S was inducible upon administration of recombinant G-CSF using a mini-osmotic pump in non-transgenic recipients, suggests that an elevated level of this cytokine is important for the inhibition of allo-resistance. Thus, G-CSF was found to play a role in allogeneic resistance to marrow grafts and the G-CSF-transgenic mice provide a useful model to study the inhibition of the resistance. The inhibition of allo-resistance may be useful in preparing allogeneic bone marrow chimeras in both experimental and clinical settings.     

Delayed effects of long-term administration of granulocyte colony-stimulating factor to mice

We studied the effects of chronic administration of granulocyte colony-stimulating factor in nonmobilizing doses to mice. Over 18 months of the study, 55% animals of the treatment group died of unknown cause, blood diseases and tumors were found in 20% mice, and in 5% animals pathological changes were absent. Control mice had no diseases (normal values of total and differential leukocyte count). The diagnoses made over the first 7 months mainly included myeloproliferative diseases. Solid tumors were found at later terms. Suppurative inflammation at the site of injection was observed in all mice after 3-month treatment with granulocyte colony-stimulating factor. Our results indicate that chronic administration of granulocyte colony-stimulating factor in low doses leads to the development of etiologically different tumors and sharply reduced animal life span. The use of granulocyte colony-stimulating factor during allogeneic transplantation of hemopoietic stem cells can be hazardous for donors. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 568–573, May, 2008     

Binding of lysozyme to lipopolysaccharide suppresses tumor necrosis factor production in vivo.

Endotoxin (lipopolysaccharide [LPS]) released during gram-negative bacterial infection induces varieties of cytokines which directly and/or indirectly cause shock, disseminated intravascular coagulation, and death. We previously showed that lysozyme (LZM) was an LPS-binding protein and inhibited various immunomodulating activities of LPS. In this study, we examined the effect of LZM on the LPS-triggered septic shock model induced by carrageenan treatment and assessed by tumor necrosis factor production. The data presented in this report strongly suggest that LZM-LPS complex formation completely abrogates tumor necrosis factor production and the mortality caused by LPS and that LZM may be useful for the treatment of endotoxin shock.     

Proliferation, macrophage colony-stimulating factor, and macrophage colony-stimulating factor-receptor expression of alveolar macrophages in active sarcoidosis.

Alveolar macrophages (AM) in sarcoidosis display an enhanced mitotic activity. Immunocytochemical detection of the proliferation-associated Ki-67 antigen revealed significant increase in the number of proliferating AM in active sarcoidosis as compared with inactive stages of disease. Macrophage proliferation may provide an additional marker of disease activity. Since growth of macrophages is regulated by hematopoietic growth factors, we examined the expression of macrophage colony-stimulating factor (M-CSF), granulocyte M-CSF, and interleukin-3 by bronchoalveolar lavage cells in active sarcoidosis. Expression of granulocyte M-CSF or interleukin-3 genes could not be detected. AM in active sarcoidosis displayed M-CSF RNA to a comparable level like normal AM. They differed, however, in about 50% of cases analyzed, from normal AM by an enhanced level of c-fms proto-oncogene (M-CSF-receptor) expression. The enlarged proportion of proliferating AM in active sarcoidosis may be the result of an increased influx of strongly fms expressing macrophage precursors into the alveoli and autostimulation of macrophages by M-CSF.     

Granulocyte-macrophage colony-stimulating factor modulates lipopolysaccharide (LPS)-binding and LPS-response of human macrophages: inverse regulation of tumour necrosis factor-alpha and interleukin-10

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a well-known stimulus for the activation, differentiation and survival of monocytes (MO). Up to now most investigations focused on the short-term effects of GM-CSF. In this study we investigated the effects of GM-CSF on the long-term differentiation of human MO in the presence of serum. We found that MO-derived macrophages (Mphi) cultured with serum plus GM-CSF (GM-Mphi) were different from control Mphi (SER-Mphi) in terms of lipopolysaccharide (LPS)-stimulated cytokine release: GM-Mphi showed an increased tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production, especially at lower LPS concentrations, but the secretion of IL-10 was diminished. In addition, GM-Mphi secreted TNF-alpha but not IL-6 and IL-10, spontaneously. The spontaneous TNF-alpha production was not due to LPS contamination as it could not be blocked by anti-CD14 antibody. Flow cytometry revealed, however, that the receptor for LPS, CD14, was up-regulated on GM-Mphi and those Mphi released twice as much soluble CD14 into the supernatant as compared with SER-Mphi. The higher CD14 expression also resulted in an enhanced LPS-binding capacity of GM-Mphi. Furthermore, the LPS-response of GM-Mphi could only be blocked by about fourfold higher concentration of anti-CD14 antibody compared with SER-Mphi. In summary, GM-CSF promotes the generation of a pro-inflammatory type of Mphi in two different ways: first, the down-regulation of autocrine IL-10 production increases the release of cytokines such as IL-6 and TNF-alpha and second, the up-regulation of membrane and soluble CD14 expression leads to a higher sensitivity towards LPS-stimulation.     

Recombinant Leishmania major secreting biologically active granulocyte-macrophage colony-stimulating factor survives poorly in macrophages in vitro and delays disease development in mice

Leishmania is an intracellular pathogen that replicates inside macrophages. Activated macrophages produce a specific subset of cytokines that play an important role in the control of Leishmania infections. As part of our interest in developing suicide parasites that produce abortive infections for the purposes of vaccination, we engineered recombinant Leishmania major strains producing biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF). We showed that GM-CSF is being produced in the phagosomes of infected macrophages and that it can be detected in the culture supernatants of both infected macrophages and extracellular parasites. Our data support the notion that GM-CSF secreted by both developmental forms of recombinant L. major can activate macrophages to produce high levels of proinflammatory cytokines such as interleukin-1beta (IL-1beta), IL-6, and IL-18 and various chemokines including RANTES/CCL5, MIP-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL2, and MCP-1/CCL2, which enhance parasite killing. Indeed, GM-CSF-expressing parasites survive poorly in macrophages in vitro and produce delayed lesion development in susceptible BALB/c mice in vivo. Selective killing of intracellular Leishmania expressing cytokine genes capable of activating cellular responses may constitute a promising strategy to control and/or prevent parasitic infections.     

Transfer of antigen from macrophages to lymphocytes: II. Immunological significance of the transfer of lipopolysaccharide

The immunological significance of lipopolysaccharide (LPS) transfer from macrophage into lymphocytes was studied in an in vitro system in which the lymphocytes were incubated during 24–72 hours with macrophages which had previously ingested S. enteriditis LPS (during 1 hour).

Our results show that the in vitro immune response to LPS requires co-operation of lymphocytes with macrophages.

After 24–72 hours of contact of the lymphocytes with macrophages containing ingested antigen, we observed within lymphocyte population a time dependent increase of the number of LPS-binding cells. This result was obtained by means of two different methods: rosettes formation and autoradiography. Autoradiographies performed on thick and thin sections showed that after contact with macrophages which had ingested ¹⁴C-LPS, most rosettes forming cells (RFC) exhibited radioactivity.

All these findings strongly argue in favour of the idea that LPS transfer from macrophages to lymphocytes is highly relevant for specific antibody synthesis by lymphocytes.

     

In vivo role of macrophages in transmission of dengue virus-induced suppressor signal to T lymphocytes

Dengue type 2 virus (DV)-induced suppressor T cells (Ts1) produce a soluble suppressor factor (SF) which stimulates a subpopulation of T lymphocytes (Ts2) to produce prostaglandin which suppresses DV-specific IgM antibody plaque forming cells (PFC) in vivo and in vitro. The present study was undertaken to investigate the intermediary role of macrophages in transmission of signal from Ts1 to Ts2. The SF is adsorbed on live or heat-killed mouse peritoneal macrophages and transmits DV-specific PFC in spleen after i.p. or i.v. inoculation in DV-primed mice. The suppression is antigen-specific. SF or SF-adsorbed heat-killed macrophages failed to transmit suppression in silica-treated macrophage-depleted mice when injected i.p., while SF-adsorbed live macrophages could transmit suppression. In macrophage-depleted mice suppression could be transmitted by SF-adsorbed heat-killed macrophages on i.v. inoculation. It was therefore concluded that live macrophage-killed cells are also essential for transmission of suppressor signal by SF from Ts1 to Ts2 in vivo.     

Bone marrow progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor versus macrophage colony-stimulating factor differentiate into macrophages with distinct tumoricidal capacities

Bone marrow-derived cells from C3H/HeJ mice were cultured in the presence of recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) or highly purified murine macrophage colony-stimulating factor (CSF-1) for 7 days. Following this 7-day culture period, mature macrophages were harvested and replated at precise densities in the absence of exogenous rGM-CSF or CSF-1, and assayed in a two-signal tumoricidal assay. Cultures were stimulated with medium only or with combinations of recombinant interferon-gamma (rIFN-gamma) as the "priming" signal, and/or butanol-extracted lipopolysaccharide (But-LPS) as the "triggering" signal for 24 hr. At this time, 51Cr-labeled, P815 tumor target cells were added, and the percent tumor cell cytotoxicity was determined after 16 hr. Macrophages derived under the influence of rGM-CSF exhibited significant tumoricidal capacity with medium alone (16 +/- 5%). The addition of "priming" signal only (i.e., rIFN-gamma, 10.0 U/ml) significantly increased tumoricidal capacity to 31 +/- 9%. Treatment with But-LPS alone did not alter the basal tumoricidal activity of rGM-CSF-derived macrophages. Combinations of rIFN-gamma (10.0 U/ml) and But-LPS (0.5-5.0 micrograms/ml) generated highly tumoricidal macrophages (50-60% tumor cell cytotoxicity). In contrast, medium-treated CSF-1-derived macrophages exhibited a significantly lower basal level of tumor cytotoxicity (6 +/- 3%). Unlike rGM-CSF-derived macrophages, treatment of CSF-1-derived macrophages with high concentrations of rIFN-gamma alone did not increase significantly the level of cytotoxicity above that of medium-treated cultures. However, CSF-1-derived macrophages responded to the highest concentrations of But-LPS (5.0 micrograms/ml) to increase tumoricidal activity from 6 +/- 3% to 17 +/- 5%. Optimal tumoricidal activity (44 +/- 17%) was observed when CSF-1-derived macrophages were treated simultaneously with high concentrations of both rIFN-gamma and But-LPS. Thus, macrophages derived from bone marrow progenitors in either rGM-CSF or CSF-1 exhibited tumoricidal capacities that differed in basal activity as well as in their requirements for and sensitivities to "priming" and "triggering" signals.