Resident Mouse Macrophages Research Articles

Investigation on the role of temperature in macrophage responses

Abstract This study investigated temperature-associated changes in macrophage activation and metabolism across species. Macrophages were isolated from C57BL/6J mice, broiler chicken spleens, and rainbow trout head kidneys, treated with lipopolysaccharide (LPS) and nitric oxide (NO) responses measured. Mouse macrophage NO production was similar at 37°C (normal body temp.) and 35°C. However at 33°C and 28°C responses dropped notably. At 39°C, Mouse bone marrow-derived macrophages (BMDM) and mouse cell line, RAW264.7, showed higher NO production versus 37°C. In chicken splenic macrophages (CSM) LPS-induced NO responses were similar at 41°C (normal for avian) 37°C, and 39°C. A mild fever of 42°C had a large stimulatory effect on NO production compared to 41°C. During infection, trout induce fever by swimming to warmer water. We found NO responses in LPS-treated head kidney macrophages to be higher at 15, 28, and 37°C than at 4°C. Total protein synthesis was evaluated via fluorescence. Without LPS stimulation, peritoneal resident mouse macrophages (PRM) from young mice showed similar protein synthesis at 37°C and 39°C. In both young and aged LPS-stimulated PRM, significantly higher protein synthesis was observed at 39°C compared to 37°C, though the increase was smaller in aged PRM. However, this effect was not uniform across all tissues. LPS stimulated young (BMDM) showed an increased protein synthesis at 37°C but cells from aged did not. A further increase in LPS stimulation was observed at 39°C. Consistent with fever increasing protein synthesis, LPS-stimulated CSM and chicken cell line HTC, showed higher protein synthesis at 42°C compared with 41°C.

TSP‐1 Binds Thr92 Phosphorylated CD36 in a Calcium Dependent Manner and the Ligands of CD36 Regulate the Phosphorylation of Thr92 in Mouse Macrophages

CD36 is an 88 kD transmembrane glycoprotein expressed on many cell types, including platelets, macrophages and endothelial cells. Our lab and others showed that CD36 is required for the anti‐angiogenic property of Thrombospondin‐1(TSP‐1), an extracellular matrix protein. Threonine 92 (Thr92) of CD36 is an important regulator of the binding of TSP‐1. Thr92 of CD36 is within a consensus protein kinase C (PKC) phosphorylation site and can be phosphorylated by PKC in vitro. The few published studies on CD36 phosphorylation in platelets suggest that the phosphorylated form, which has low affinity to TSP‐1, may be the default state. To study the phosphorylation of Thr92 of CD36, we cloned a 111bp CD36 fragment (FP111) that encodes amino acids 81 to 117. We showed that the WT but not T92A FP111 could be phosphorylated by PKC in vitro. FP111 binds TSP‐1 in vitro in a calcium dependent manner and the phosphorylation of FP111 enhances the binding of TSP‐1. Thr92 of CD36 in both elicited and resident mouse macrophages is phosphorylated, at least in part. Moreover, the phosphorylation level of CD36 in mouse macrophages can be regulated by CD36 ligands. Ectophosphorylation of CD36 is a novel mechanism of regulation and may impact CD36‐dependent functions. Determination of the effect and the regulation of CD36 phosphorylation will improve our understanding of the regulation of CD36 dependent functions, such as angiogenesis.

Posttranscriptional regulation of TNFα expression via eukaryotic initiation factor 4E (eIF4E) phosphorylation in mouse macrophages

Resident mouse macrophages secrete Tumor necrosis factor α (TNFα) upon challenge with LPS. The production of TNFα is controlled not only at the transcription of the gene, but also by strong posttranscriptional regulation. When macrophages are stimulated with LPS different signal transduction pathways become activated. Here we show that the combination of the 2 kinases p38 and MEK and presumably ERK1/2 regulate translation of TNFα, through the downstream kinase Mnk1. TNFα production is inhibited in a concentration-dependent manner by CGP57380 (Mnk1 inhibitor). The corresponding mRNA results show that the inhibition targets posttranscriptional regulation and is paralleled by inhibition of the phosphorylation of eukaryotic initiation factor 4E (eIF4E). Unexpectedly, the activation/inhibition of MAPKAP kinase-2 (MK2) does not parallel TNFα production, arguing against a direct/immediate role for this kinase. On the basis of the present and previous results we propose that ARE-containing TNFα mRNA requires phosphorylation of eIF4E for initiation of translation.

Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms.

The elimination of serum or of serum-derived macromolecules that supplant the fetal calf serum requirement from Leishmania culture media could decrease costs and improve the feasibility of large-scale production of well-defined parasite material. We report a completely defined medium, without serum-derived protein and/or macromolecules as a serum substitute, of common, available, and inexpensive constituents that can be used in place of serum-supplemented media for the continuous in vitro cultivation of promastigote forms of various Leishmania species. Typical promastigote morphology was observed in Giemsa-stained smears, regardless of the strain analyzed. Electrophoretic analysis showed that the proteinase patterns of aserically grown promastigote forms were similar to those obtained in serum-supplemented RPMI 1640 medium for all Leishmania studied. Similar antigenic profiles were recognized in immunoblots by sera from hosts with visceral or cutaneous leishmaniasis after growing promastigotes in the two different culture media. For parasites causing both cutaneous and visceral leishmaniasis, the absence of serum and macromolecules in the culture medium did not markedly change their in vitro infectivity for resident mouse macrophages and their virulence in animals compared with parasites cultivated in nondefined medium. Serum-free technology will be increasingly important in providing stability and reproducibility as research using promastigote moves closer to therapeutic applications.

In vitro life cycle of pentamidine-resistant amastigotes: stability of the chemoresistant phenotypes is dependent on the level of resistance induced.

Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana. We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 microM pentamidine, while clone LmPENT20 was resistant to 20 microM pentamidine. Resistance indexes (50% inhibitory concentration [IC50] after drug presure/IC50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection. Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine. Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987). No difference in the in vitro infectivity for resident mouse macrophages was observed between the wild-type clone (clone LmWT) and pentamidine-resistant clones. During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 microM pentamidine. In the same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC50s and index of resistance values close to those for the LmWT clone. These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance. This fact could be significant in the in vivo transmission of drug-resistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

Cyclosporin-sensitive expression of cytokine mRNA in mouse macrophages responding to bacteria

Characteristics of the cytokine response in resident mouse macrophages to certain Gram-positive and Gram-negative bacteria have been investigated by monitoring the expression of mRNA encoding interleukin-1α and -β ( IL- 1α β ) and tumor necrosis factor-α (TNF-α). Expression of these cytokine mRNAs occurred within 30–60 min. Both the flavonoid quercetin and phloretin inhibited the expression of IL- 1α β as well as TNF-α mRNA, with quercetin being more potent than phloretin and TNF-α expression somewhat more sensitive than that of IL- 1α β . Expression of all three cytokine mRNAs was also inhibited by prostaglandin E 2, with an IC 50 of > 1 μM, but not by the phosphodiesterase inhibitor pentoxifylline, although lipopolysaccharide-induced expression of TNF-α mRNA was inhibited. Down-regulation of phorbol ester-sensitive isoforms of protein kinase C had virtually no effect on the cytokine response to bacteria, and treatment of resting macrophages with phorbol ester did not cause expression of any of the cytokine mRNAs investigated. Among protein phosphatase inhibitors, cyclosporin A caused extensive inhibition of bacteria-induced expression of both IL- 1α β and TNF-α mRNA, while okadaic acid in itself caused selective induction of TNF-α, but not IL- 1α β mRNA, with a sharp peak at 0.3 μM concentration. At higher concentrations of okadaic acid, at which protein/phosphatase 2B/calcineurin would also be inhibited, the induction was completely reversed. This suggests that critical phosphorylation events, counteracted by one or more okadaic acid-sensitive protein phosphatase(s), and a dephosphorylation event carried out by a cyclosporin-sensitive protein phosphatase are both necessary for transcriptional activation of the TNF-α gene.

Cytosolic pH regulation in mouse macrophages. Characteristics of HCO3(-)-dependent mechanisms.

Mechanisms regulating cytosolic pH (pHi) in adherent resident mouse macrophages have been characterized by use of the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Na+/H+ exchange was activated after an acid load of the macrophage cytosol. However, when Na+/H+ exchange was the only pHi-regulatory mechanism operative, recovery did not proceed beyond a pHi of approx. 6.6. The mechanisms found to be operative at physiological pHi levels were alkalinizing Na(+)-dependent and acidifying Na(+)-independent Cl-/HCO3- exchangers and a H(+)-ATPase further characterized in the accompanying paper [Tapper & Sundler (1992) Biochem. J. 281, 245-250]. Acid extrusion via Na+/Cl-/HCO3- exchange was demonstrated by the dependence on external Na+ and HCO3- and on internal Cl- and by the sensitivity to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS). By monitoring pHi changes upon Cl- removal and re-addition, the pH-dependence and sensitivity to SITS were found to differ for the alkalinizing and the acidifying Cl-/HCO3- exchangers.

Tumor necrosis factor-alpha triggers antitoxoplasmal activity of IFN-gamma primed macrophages.

IFN-gamma is an important mediator of cellular resistance against microbial pathogens and tumor cells due in part to its potent capacity to activate macrophages for enhanced cytotoxicity. The present study demonstrates that TNF-alpha regulates the expression of enhanced antimicrobial activity by triggering IFN-gamma primed macrophages to kill or inhibit intracellular Toxoplasma gondii. Resident mouse macrophages stimulated with rIFN-gamma at levels up to 2500 U/ml failed to display enhanced antitoxoplasmal activity when cultured in vitro under low endotoxin conditions (less than 10 pg/ml), but were triggered by addition of small amounts of LPS (0.1 ng/ml). A similar requirement for LPS as a second signal necessary to trigger antitoxoplasmal activity was observed when IFN-gamma was administered to mice in vivo. The essential nature of this triggering step allowed us to explore the role of cytokines that act as endogenous regulators of macrophage activation. rTNF-alpha, although unable to confer antitoxoplasmal activity when used alone to treat macrophages, was capable of triggering IFN-gamma-primed macrophages cultured under low endotoxin conditions. The ability of TNF-alpha to trigger IFN-gamma-primed macrophages was blocked by rabbit anti-TNF-alpha polyclonal antisera but was not affected by polymyxin B indicating that TNF-alpha triggering was not due to contamination with LPS. Collectively, these findings demonstrate that TNF-alpha performs an important regulatory role in the expression of enhanced anti-microbial activity by IFN-gamma-primed macrophages.

Extracellular ATP induces a large nonselective conductance in macrophage plasma membranes.

Extracellular ATP in its tetra-anionic form (ATP4-) induces ion fluxes and membrane depolarization in the mouse macrophage-like cell line J774.2 and in resident mouse macrophages. We analyzed the effects of extracellular ATP4- by both patch-clamp and intracellular microelectrode techniques. Whole-cell patch-configuration membrane potential measurements on J774.2 cells revealed that ATP4- -induced depolarization occurred within 40 ms of pulsed application of ATP and was completely reversible. The depolarizations were accompanied by a dramatic increase in membrane conductance and showed no sign of adaptation to ATP over a period of 30 min. At 5 mM total ATP (ATPt) the whole-cell conductance was approximately 10 nS, and an upper limit of 20 pS for a single-channel conductance has been established. The reversal potential associated with the ATP-induced depolarization at asymmetric K+, Na+, Ca2+, and Cl- concentrations across the membrane was 0 mV. In patch-clamped cells depolarization was complete at 20 microM ATP4-, and repolarization from full depolarization occurred in approximately 5 s. In contrast, in intact cells measured by microelectrode impalement, complete depolarization occurred at approximately 2 mM ATP4- and repolarization was much slower (approximately 100 min). These findings indicate that the changes in intracellular ionic composition that occur after ATP treatment affect the rate of cell repolarization. At lower concentrations of ATP, potassium conductances modulated the depolarizing effect of ATP. ATP also depolarized mouse peritoneal macrophages, but a variant cell line (ATPR B2), derived from J774.2 cells by prolonged exposure to ATP, was insensitive to ATP. Our results provide a membrane electrophysiological description and analysis of a large nonselective plasma membrane conductance of macrophages induced by extracellular ATP.

Elementary body envelopes from Chlamydia psittaci can induce immediate cytotoxicity in resident mouse macrophages and L-cells.

Isolated, purified Chlamydia psittaci elementary body envelopes at a high multiplicity of infection (1,000:1) are capable of inducing immediate cytotoxicity in resident mouse macrophages and 929 L-cells.

Elementary Body Envelopes from Chlamydia psittaci Can Induce Immediate Cytotoxicity in Resident Mouse Macrophages and L-Cells

Elementary Body Envelopes from Chlamydia psittaci Can Induce Immediate Cytotoxicity in Resident Mouse Macrophages and L-Cells

Regulation by Fc fragments of the secretion of collagenase, PGE2, and lysozyme by mouse peritoneal macrophages.

Fc fragments of human IgG can stimulate resident mouse macrophages in culture to secret collagenase, to increase PGE2 secretion, and to decrease the secretion of lysozyme. Active synthesis and secretion were shown by the progressive accumulation of these products in the extracellular medium and inhibition of secretion by cycloheximide. A dose-dependent effect of Fc fragments was demonstrable. Brief exposure of cells to Fc fragments was sufficient to cause the macrophages to secrete collagenase and large amounts of PGE2 for prolonged periods of time, suggesting that a sustained activation rather than temporary modulation of the cells had occurred. Con A had similar effects on macrophage secretory activity. These findings indicate that proteins that bind to specific macrophage plasma membrane receptors may stimulate the secretion of products that promote the inflammatory response.