Nitric oxide-mediated protection of A. actinomycetemcomitans-infected murine macrophages against apoptosis.

We investigated apoptotic cell death in murine macrophage cell line J774.1 following Actinobacillus actinomycetemcomitans infection. Infected macrophages generally kill bacteria within phagosomes with nitric oxide (NO). Our previous study demonstrated that DNA fragmentation in infected cells increased significantly on addition of S-Methylisothiourea (SMT), a selective inhibitor of inducible NO synthetase (iNOS). The purpose of the present study was to determine the mechanism via which NO affects apoptosis of infected macrophages. J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1. The infected cells were then cultured in the presence or absence of SMT (400 microM). Culture supernatant was removed 21 h after the infection to measure LDH activity. Additionally, cellular proteins were extracted from the infected cells and measured for histone-associated DNA fragmentation and caspase-1, -3, -5, -6, -8, -9 activities. LDH activity and DNA fragmentation were significantly elevated by the infection; moreover, levels increased further on addition of SMT. Caspase activity of infected cells, particularly caspase-3, was significantly higher than that of uninfected cells. Furthermore, caspase activity increased on addition of SMT. These findings indicate that NO protects infected J774.1 cells, at least in part, against apoptotic cell death via a decrease in caspase activity.     

Involvement of caspase activation through release of cytochrome c from mitochondria in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans

We previously reported that infection with the periodontopathic bacterium Actinobacillus actinomycetemcomitans induced apoptosis in a mouse macrophage cell line J774.1. In the present study, we examined the involvement of cytochrome c and caspases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells. Following infection, the expression levels of cytochrome c, and cleaved forms of caspase-3 and caspase-9 in the cells were examined using immunoblot analysis. Cytochrome c was released from mitochondria into the cytoplasm after A. actinomycetemcomitans-infected J774.1 cells were cultured for 6 h, and caspase-3 and caspase-9 were found to be cleaved forms in the cells. Further, caspase-9 activity was markedly increased, and phosphorylated p53 was detected in the cells 30 h following infection. These results suggest that apoptosis in A. actinomycetemcomitans-infected J774.1 cells is regulated by the release of cytochrome c from mitochondria into cytoplasm and the subsequent activation of caspases through phosphorylation of p53.     

Suppressive effects of serum on the LPS-induced production of nitric oxide and TNF-alpha by a macrophage-like cell line, WEHI-3, are dependent on the structure of polysaccharide chains in LPS

The effect of serum on LPS-induced activation of a murine macrophage-like cell line, WEHI-3, was examined. Foetal calf serum strongly inhibited the production of nitric oxide (NO) and TNF-alpha by LPS-stimulated WEHI-3 cells, while it enhanced the production of both by other macrophage-like cell lines, J774.1 and BAM3, on treatment with LPS. This suppressive effect of serum on WEHI-3 cells was most remarkable when the cells were stimulated with rough-chemotype LPS, Ra LPS, Rc LPS and Rd2 LPS. Foetal calf serum also inhibited TNF-alpha production by the same cells stimulated with high concentrations of smooth-form LPS (S LPS; > 1000 ng/mL). Serum-mediated suppression was also observed for expression of the TNF-alpha gene in Rc LPS-stimulated WEHI-3 cells. This suppressive effect of FCS was most remarkable during the 1-2 h before the addition of LPS, but it was not observed when FCS was added at 1 h after the addition of LPS, suggesting dependence on the time of FCS addition to LPS-stimulated cells. No significant difference was observed in the expression of CD14 on WEHI-3 cells cultured in the presence and absence of serum, suggesting that CD14 is not involved in the serum-mediated suppression of these LPS-responses. On the contrary, FCS showed enhancing effects on the production of NO and TNF-alpha by WEHI-3 cells stimulated with low concentrations (< 100 ng/mL) of S LPS and rough mutant Salmonella minnesota Re LPS. These results suggest that the ability of FCS to suppress LPS-induced activation of WEHI-3 cells in mainly dependent on the structure of polysaccharide chains and also on the concentration of LPS employed.     

Isolation of a lipopolysaccharide (LPS)-resistant mutant, with defective LPS binding, of cultured macrophage-like cells.

Lipopolysaccharide (LPS)-resistant mutants which did not respond to LPS were isolated from a macrophage-like mouse cell line, J774.1. Unlike the parental J774.1 cells, these mutants grew even in LPS added medium as well as in normal growth medium without any morphological changes. Assay of 125I-LPS binding to the cell monolayers revealed that one of these LPS-resistant mutants (LR-9) was strikingly defective in LPS-binding activity. Scatchard plot showed that LR-9 cells lacked the high affinity binding sites which were present in J774.1. The high affinity binding was inhibited by addition of excess unlabeled LPS, lipid A, lipid IVA (tetraacyl-beta(1'-6)-linked D-glucosamine disaccharide-1,4'-bisphosphate), and lipid X (2,3-diacylglucosamine 1-phosphate) and sensitive to proteinase K. LPS enhanced O2- generation and the release of arachidonic acid in J774.1 cells but not in LR-9 cells. Other stimulants such as zymosan and 12-O-tetradecanoylphorbol 13-acetate, however, induced the release of arachidonic acid in LR-9 cells as well as in J774.1 cells. LPS-photocross-linked assay allowed the identification of 65- and 55-kDa LPS-binding proteins in the membrane fraction of J774.1 cells. Both of the bands were not detectable in that of LR-9 cells and disappeared by competing with unlabeled LPS or lipid X. These results show that one or both of the two LPS-binding proteins might relate to the specific membrane receptor for LPS.     

Diphosphoryl lipid A derived from the lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 is a potent competitive LPS inhibitor in murine macrophage-like J774.1 cells

Abstract Pentaacyl diphosphoryllipid A derived from the nontoxic lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 (RsDPLA) did not induce tumour necrosis factor-α nor interleukin-6 release in the murine macrophage-like cell line J774.1. However, it effectively inhibited the induction of these two cytokines by LPS of Salmonella minnesota Re mutant R595 (ReLPA) in a concentration-dependent manner. Maximal inhibition and half-maximal inhibition occured when the ReLPS to RsDPLA mass ratio was 1:30 and 1:1, respectively. A binding study was performed in the presence of serum to determine whether RsDPLA is competing with ReLPS for LPS binding sites on J774.1 cells. This assay allows the determination of LPS binding to J774.1 cells via a mechanism involving CD14, a receptor for complexes of LPS with LPS binding protein (LBP), and its possible inhibition. The results show that RsDPLA strongly inhibits the binding of ¹²⁵I-labelled ReLPS to J774.1 cells. Maximal and one-half maximal inhibition of binding occured when the ReLPS to RsDPLA mass ratios were 1:2.5 and 1:0.5, respectively. It was found that the inhibition of binding by RsDPLA was much stronger than that by unlabelled ReLPS. These results suggest that RsDPLA is competing with ReLPS for CD14-dependent recognition of LPS on J774.1 cells.     

Induction of ornithine decarboxylase in macrophages by bacterial lipopolysaccharides (LPS) and mycobacterial cell wall material

Lipopolysaccharide from $\text{E. Coli}$ (LPS) and BCG cell walls (BCGcw) are recognized immunoadjuvants that directly stimulate some macrophage functions. The macrophage cell line J774.1 and peritoneal exudate cells (PEC) from mice can be stimulated by LPS or other adjuvants $\text{in vitro}$ to synthesize and release protein factor(s) that activate thymus-derived lymphocytes. We have utilized J774.1 cells and PEC to demonstrate that an increase in ornithine decarboxylase (ODC) activity is a marker of early biochemical changes in adjuvant-stimulated macrophages. BCGcw and LPS increased ODC within 2 hours in J774.1 cells as well as murine peritoneal exudate macrophages. Maximal increases in ODC were detected 4 hours after the addition of adjuvants to J774.1 cells. The marked increases (12–23 fold) in ODC observed with BCGcw (20 μg/ml) did not appear to involve an effect on cell proliferation which was suppressed by this adjuvant. Cycloheximide inhibited the induction of ODC by LPS and BCGcw in the macrophage cell line. Evidence that the induction of ODC may be promoted by an increase in cyclic AMP was provided by experiments demonstrating that prostaglandin E₁ (PGE₁) and 8-bromo-adenosine-3′:5′-monophosphate (8Br-cyclic AMP) can mimic the effects of LPS and BCGcw in J774.1 cells. These observations indicate that one of the early biochemical changes in macrophages promoted by adjuvants is an induction of ODC.     

6]-Gingerol inhibits nitric oxide synthesis in activated J774.1 mouse macrophages and prevents peroxynitrite-induced oxidation and nitration reactions

Reactive nitrogen species (RNS), such as nitric oxide (NO) and its derivatives, e.g. peroxynitrite (ONOO-), have been proposed as being able to influence signal transduction and cause DNA damage, contributing to carcinogenic processes. In this study, the effect of [6]-gingerol, a pungent phenolic compound present in ginger (Zingiber officinale Roscoe), on NO synthesis in lipopolysaccharide (LPS)-activated J774.1 macrophages was tested, and the protective ability of this compound against peroxynitrite-mediated oxidation and nitration reactions were evaluated. [6]-Gingerol exhibited dose-dependent inhibition of NO production and significant reduction of inducible NO synthase (iNOS) in LPS-stimulated J774.1 cells. Moreover, [6]-gingerol effectively suppressed peroxynitrite-induced oxidation of dichlorodihydrofluorescein, oxidative single strand breaks in supercoiled pTZ 18U plasmid DNA, and formation of 3-nitrotyrosine in bovine serum albumin (BSA) and J774.1 cells. Our results indicate that [6]-gingerol is a potent inhibitor of NO synthesis and also an effective protector against peroxynitrite-mediated damage.     

Ornithine-containing lipids stimulate CD14-dependent TNF-alpha production from murine macrophage-like J774.1 and RAW 264.7 cells

The ornithine-containing lipids (OL)-induced cytokine production pattern in macrophage-like J774.1 and RAW 264.7 cells was different from that in the peritoneal macrophages previously reported. OLs, as well as lipopolysaccharide (LPS) of Escherichia coli, strongly induced tumor necrosis factor (TNF) alpha but not interleukin (IL)-1beta in J774.1 cells. In the RAW cells, IL-1beta, TNF-alpha and prostaglandin E(2) were strongly induced by the OLs and LPS. OL- and serine-glycine-containing lipid (SGL)-induced TNF-alpha production in J774.1 and RAW 264.7 cells required serum. However, in CD14-deficient LR-9 cells, TNF-alpha was not induced by the OLs in the presence or absence of serum. OLs and a SGL almost completely inhibited the binding of (125)I-LPS to J774.1 cells. These results suggested that OLs and SGL activate macrophages via the CD14-dependent pathway.     

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.     

LPS-or 8Br-cyclic AMP-induced production of T cell-activating factor(s) in macrophage tumor cell line J774.1.

The macrophage tumor cell line J774.1 replaced the function of normal macrophages in the induction of polyclonal killer T cells with 2-mercaptoethanol. J774.1 does not normally release soluble factor(s) which we have shown to be responsible for the differentiation of T cells to killer T cells. However, stimulation of J774.1 with LPS induced soluble factor(s) for T cell activation. An optimum concentration of LPS for the production of soluble factor(s) was 1 to 10 microgram/ml, which completely inhibited growth of the tumor cells. The production of soluble factor(s) was observed within 6 hr after LPS stimulation and reached its maximum level at 24 hr. Incubation of the cell line with 8Br-cyclic AMP and theophylline induced soluble factor(s), suggesting that LPS stimulation induced an increase in intracellular cyclic AMP which leads to the synthesis of soluble factor(s).     

Participation of CD14 in the Phagocytosis of Smooth-Type Salmonella typhimurium by the Macrophage-Like Cell Line,J774.1

The role of CD14 in the phagocytosis and killing of microorganisms was investigated using macrophage-like cell lines, CD14-positive J774.1 cells and CD14-negative mutant J7.DEF3 cells derived from J744.1 cells. The cells were infected with Salmonella typhimurium organisms of the smooth (S)-form LT2, mutant rough (R)-form TV148 or Staphylococcus aureus 248βH. At 30 or 180 min incubation, the cells were washed and disrupted. Colony-forming units (CFUs) liberated from the disrupted cells were determined by quantitative cultivation, and the phagocytic index and killing rate were calculated. Both the phagocytic index and killing rate of J774.1 cells against LT2 organisms were greater than those of J7.DEF.3 cells. However, the index and rate of J774.1 cells against TV148 and 248βH organisms were similar to those of the J7.DEF.3 cells. The phagocytosis of LT2 organisms by J774.1 cells was partially inhibited by S-form LPS (S-LPS) and anti-CD14 antibody, but not by R-chemotype LPS (R-LPS). These results suggest that CD14 participates in the phagocytosis of S-form Salmonella.     

l-NAME inhibits tumor cell progression and pulmonary metastasis of r/m HM-SFME-1 cells by decreasing NO from tumor cells and TNF-α from macrophages

Highly metastatic ras/myc-transformed serum-free mouse embryo (r/m HM-SFME-1) cells were injected subcutaneously to mice and the effects of Nω-nitro-l-arginine methyl ester (l-NAME) on the tumor progression and pulmonary metastasis were investigated. In addition, production of nitric oxide (NO), matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-α) in the tumor cells and in a mouse macrophage-like cell line, J774.1 cells, was analyzed. The increase in footpad thickness was significantly smaller in the mice which were fed the l-NAME containing water (4.24 ± 0.39 mg/day/mouse). The number of the tumor cells metastasized to the lungs was smaller in the l-NAME treated mice, although statistical significance was not found. Co-treatment of r/m HM-SFME-1 cells with interferon-gamma (IFN-γ; 100 U/ml) and lipopolysaccharide (LPS; 0.5 μg/ml) significantly enhanced NO production, and the presence of l-NAME at 1 mM significantly decreased this response. In r/m HM-SFME-1 cells, MMP-2 was undetectable and MMP-9 was also very little in the basal level, and both MMPs were unaffected by the IFN-γ and/or LPS treatments, not to mention by the l-NAME treatment. In J774.1 cells, any treatment including LPS appeared to enhance MMP-9 production, however, this upregulation was not inhibited by the additional presence of l-NAME. Production of TNF-α by J774.1 cells was markedly enhanced with LPS treatment, and this enhancement was significantly reduced in the presence of l-NAME. These results indicate that the inhibitory effects of l-NAME on the tumor cell progression and pulmonary metastasis could be due to suppression of NO from tumor cells and TNF-α from macrophages (Mol Cell Biochem, 2007). Hideaki Yamaguchi and Yumi Kidachi contributed equally to this work.     

The significance of the hydrophilic backbone and the hydrophobic fatty acid regions of lipid a for macrophage binding and cytokine induction

Abstract Natural partial structures of lipopolysaccharide (LPS) as well as synthetic analogues and derivatives of lipid A were compared with respect to inhibit the binding of ¹²⁵I-labelled Re-chemotype LPS to mouse macrophage-like J774.1 cells to induce cytokine-release in J774.1 cells. LPS, synthetic Escherichia coli -type lipid A (compound 506) and tetraacyl percursor Ia (compound 406) inhibited the binding of ¹²⁵I-LPS to macrophage-like J774.1 cells and induced the release of tumor ncerosis factor α (TNFα) and interleukin 6 (IL-6). Deacylated R-chemotype LPS preparations were completely inactive in inhibiting binding and in inducing cytokine-release. Among tetraacyl compounds, the inhibition-capacity of LPS-binding was in decreasing order: PE-4 ( α -phosphonooxyethyl analogue of 406)>406⪢>404(4′-monophosphoryl partial structure of 406)>405 (1-monophosphoryl partial structure of 406). In the case of hexaccyl preparations, compounds 506, PE-1 (α-phosphonooxyethyl analogue of 506) and PE-2 (differing from PE-1 in having 14:0 at positions 2 and 3 of the reducing GlcN) inhibited LPS-binding and induced cytokine release equally well, whereas preparation PE-3 (differing from PE-2 in containing a β-phosphhonooxyethyl group) showed a substantially lower capacity in binding-inhibition and cytokine-induction. The conclusion is that chemical changes in the hydrophilic lipid A backbone reduce the capacity of lipid A to bind to cells, whereas the number of fatty acids determines the capacity of lipid A to activate cells. These results indicate that the bisphosphorylated hexosamine backbone of lipid A is essential for specific binding of LPS to macrophages and that the acylation pattern plays a critical role for LPS-promoted cell activation, i.e. cytokine induction.     

Induction of ornithine decarboxylase, RNA, and protein synthesis in macrophage cell lines stimulated by immunoadjuvants

Early biochemical changes associated with adjuvant stimulation of macrophage protein synthesis were studied using two murine macrophage cell lines, PU5-1.8 and J774.1. An induction of ornithine decarboxylase (ODC) was detected 2 hours after exposure of PU5-1.8 and J774.1 cells to two crude immunoadjuvants, BCG cell walls (BCGcw) and lipopolysaccharides from Escherichia coli (LPS). The chemically defined immunoadjuvant glycopeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDPL) also promoted an increase in ODC activity at 2 hours that was maximal after 4 hours, while little or no effect was observed with the D-alanyl analog (MDPD) that is devoid of adjuvant activity. The increase in ODC activity promoted by BCGcw in PU5-1.8 and J774.1 cells returned toward control levels by 6 to 8 hours. BCGcw also stimulated RNA and protein synthesis which remained elevated for at least 24 hours and was associated with a decrease in DNA synthesis and cell proliferation. ODC induction by BCGcw and MDPL was enhanced by the addition of PGE2 in both cell lines. Indomethacin slightly depressed the magnitude of ODC stimulation by BCGcw in J774.1 cells but failed to alter the response of PU5-1.8 cells. Additional observations indicated that the induction of ODC by BCGcw in both cell lines was preceded by an activation of cyclic AMP-dependent protein kinase. These observations suggest that a cyclic AMP-mediated induction of ODC may be an early biochemical marker of adjuvant stimulation in macrophages.     

Anti-inflammatory metabolite production in the gut from the consumption of probiotic yogurt containing Bifidobacterium animalis subsp. lactis LKM512

There is little evidence for a relationship between probiotic metabolites and host cytokine production. We investigated in the present study the possibility that anti-inflammatory metabolites can be produced in the gut by LKM512 yogurt consumption by using murine macrophage-like J774.1 cells and extracts prepared from the feces of elderly volunteers. These volunteers' acute inflammation had been inhibited by LKM512 yogurt consumption in a previous test. The tumor necrosis factor (TNF)-alpha production elicited in J774.1 cells stimulated by lipopolysaccharide (LPS) and in the fecal extracts obtained during the period of LKM512 yogurt consumption was significantly decreased (p<0.05) than the pre-consumption baseline level. These findings and previous data enable us to conclude that intestinal bacterial metabolites produced by LKM512 yogurt consumption contributed to suppressing the inflammatory cytokine produced by macrophages and that one of the anti-inflammatory metabolites in the fecal extracts was likely to have been a polyamine.     

Apoptotic Changes Preceding Necrosis in Lipopolysaccharide-Treated Macrophages in the Presence of Cycloheximide

Apoptotic changes occurred specifically in a macrophage-like cell line, J774.1, treated with lipopolysaccharide (LPS) and cycloheximide (CHX) prior to the release of lactate dehydrogenase (LDH). The addition of 100 ng/ml LPS and 10 μg/ml CHX induced both the formation of DNA nicks and elevation of caspase-3-like activity (DEVDase) after 75 min, and then the cleavage of poly(ADP-ribose) polymerase (PARP) into 28-kDa fragments, formation of apoptotic bodies, and DNA ladder formation. These apoptotic changes were reversible until 60 min, however, later than 75 min after LPS and CHX addition, the apoptosis proceeded normally even on extensive washing of the macrophages, which removed the LPS and CHX. These results suggest that there is a “point of no return” in the apoptotic processes in macrophages induced by LPS and CHX and that DNA nicks and activation of DEVDase are critical for these processes.     

Increased apoptosis during the early phase of experimental paracoccidioidomycosis as a phenotypic marker of resistance

Paracoccidiodomycosis (PCM) is a systemic mycosis that presents a wide spectrum of clinical manifestations caused by Paracoccidiodes brasiliensis. The experimental murine model has been used to approach the disease with susceptible and resistant mouse strains that reproduce most of the main human immunological features. We investigated whether the pattern of apoptosis of peritoneal cells from two polar strains of mice after infection with P. brasiliensis could be associated with the susceptibility or resistance to this pathogen. Apoptosis of A/J mouse cells (resistant), cultured in the presence or absence of LPS as stimuli, was observed as early as on the first day of infection. Cells from the infected susceptible strain BALB/c did not exhibit apoptosis in absence of LPS and persistently at a lesser degree than that observed in resistant mice. The apoptosis induced by the infection in resistant mice was not due to nitric oxide, since its blockage either in vitro or in vivo did not revert it. Analysis of additional strains of polar susceptibilities to PCM assured the dissociation of NO production and apoptosis. Interestingly, IL-6 and IL-10 were secreted in high amounts, by BALB/c cells and might be involved in shielding cells from apoptosis induced by P. brasiliensis. Furthermore, IFNgamma(-/-) mice did not show apoptosis of peritoneal cells while the Wt controls presented levels similar to those of A/J strain that secreted high amounts of IFNgamma and IL-1beta. The expression of Fas was increased in both strains and in Wt mice, whereas FasL was decreased in the susceptible strain and not significantly modulated in TNFRI and IFNgamma KO mice. These results suggest that apoptosis might be a mechanism of control of engagement of cells that could otherwise contribute to the susceptible phenotype observed in some strains of mice.     

Essential role of protein kinase C zeta in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP

Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) zeta was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCzeta in SASH1 cells, myristoylated PKCzeta peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway.     

Control of lipoprotein lipase secretion in mouse macrophages

The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells.