Effects of recombinant human macrophage colony-stimulating factor on atherosclerotic lesions established in the aorta of high cholesterol-fed rabbits.

Anti-atherosclerotic effects of human macrophage colony-stimulating factor were investigated using rabbits fed a high cholesterol diet. Rabbits fed a diet containing 2% cholesterol for 59 days developed hyperlipidemia and atheromatous aortic plaques. They were then administered 80 microg/kg/day of either macrophage colony-stimulating factor or human serum albumin, as a control, for the next 12 weeks. Compared with the control group, rabbits treated with macrophage colony-stimulating factor had significantly fewer plaques on the inner surface of the thoracic and abdominal aortae, and half the sectional area of thickened intima in the aortic arch, as well as in the thoracic and abdominal aortae. Macrophage colony-stimulating factor also decreased the cholesterol content of the atherosclerotic lesions. Serobiochemical analyses revealed that macrophage colony-stimulating factor increased the levels of high density lipoprotein-cholesterol significantly, without influencing other lipid parameters such as the level of low density lipoproteins. The effects of macrophage colony-stimulating factor were evident until the fourth week of drug injection, at which time anti-human macrophage colony-stimulating factor antibodies were clearly induced in the serum. These results indicate that exogenously administered macrophage colony-stimulating factor suppresses atherosclerotic lesions induced by a high cholesterol diet by activating lipid metabolism in vivo.

[The trend of the drug eruptions in the last fifteen years].

The drug eruptions are known to often become more severe by the readministration of causative drugs. It is an important theme to prevent the relapse of the drug eruptions. We have been monitoring drug adverse reactions at our hospital since October, 1980. We divided fifteen years from October, 1980 to September, 1995 into three periods; the first period (Oct., 1980-Sep. 1985), the second period (Oct., 1985-Sep., 1990), and the third period (Oct., 1990-Sep., 1995), and discussed the trend of the drug eruptions appeared among these three periods. The number of the drug eruptions increased. But the proportion to the total drug adverse reactions and to the number of patients slightly decreased. The eruptions in women much increased and in the patients of forties or older generations also increased. But in patients of thirties or younger generations decreased. While nonsteroidal anti-inflammatory drugs (NSAIDs) other than pyrines, antibiotics other than penicillins and cephalosporins and drugs affecting the cardiovascular system and the metabolism tend to increase, pyrines, penicillins, iodic and biliary contrast media tend to decrease. The incubation period before the eruption appeared is less than three days in most antibiotics and anti-inflammatory drugs. But it is more than four days in most drugs for chronic diseases. Other symptoms such as nausea, fever and liver dysfunction were shown in 9.2% of the drug eruptions. In 8.9% of the drug eruptions a relapse of allergic reactions included eruptions were also found. In some cases the drug eruptions exacerbate by re-administration of beta-lactam antibiotics. In the case of administration of drugs, it is necessary to pay attention to dermatitis caused by the drugs. And we recognized the importance of the system for the prevention of the relapse of drug eruptions including injections.

Effects of macrophage colony-stimulating factor (M-CSF) on protease production from monocyte, macrophage and foam cell in vitro: a possible mechanism for anti-atherosclerotic effect of M-CSF.

M-CSF is a growth factor that stimulates proliferation and differentiation of monocyte/macrophage-lineage cells. In our previous studies, M-CSF regresses atherosclerotic lesions preformed in aorta of high cholesterol-fed rabbit. Immunohistochemical analysis indicated that extracellular matrix (ECM), such as collagen, was especially eliminated in the intima of atherosclerotic lesion. To define the collagen-lowering potential of M-CSF, we have studied the effects of M-CSF on production of collagen-degrading proteases, such as MMP-1, -9 and urokinase in vitro. Monocytes freshly isolated from human peripheral blood produced MMP-9, but not urokinase, and M-CSF enhanced MMP-9 production. Macrophages were prepared by culturing monocytes for 10 days in the presence or absence of M-CSF, and protease production was assayed. M-CSF augmented production of MMP-9 and urokinase in a dose-dependent manner. M-CSF also enhanced MMP-1 production of macrophages, but not significantly. Foam cells were prepared by culturing macrophages in the presence of acetyl LDL, and protease production from these cells were also elevated by M-CSF. These results suggest that M-CSF exogenously administered in atherosclerotic rabbits might regress the thickened intima by activating macrophages to degrade collagen accumulated in the lesion.

Monocyte proliferation induced by modified serum is associated with endogenous M-CSF production: evidence for involvement of a signalling pathway via scavenger receptors.

It was found that human serum stored for 2 months at 4 degrees C (modified serum) induced monocyte proliferation and simultaneous macrophage colony stimulating factor (M-CSF) production by these cells in vitro. Cell number, estimated by DNA content, doubled after 10 days in culture in the presence of modified serum, while it decreased in culture with freshly thawed control serum. As the addition of more than 2.5 ng/ml of recombinant M-CSF significantly supported monocyte survival/proliferation, cells were cultured for 10 days in medium supplemented with control serum, and endogenous M-CSF production was investigated by enzyme-linked immunosorbent assay. M-CSF concentration in the supernatants was 15-30 ng/ml after 10 day in culture with modified serum, a level that might be sufficient for monocyte proliferation. The modified serum induced M-CSF from freshly isolated monocytes, while M-CSF was hardly detected in cultures supplemented with control serum. Assay for peroxidized lipid and agarose gel electrophoresis demonstrated that the modified serum contained more oxidized low density lipoproteins (LDL) than the control serum. Ligands of scavenger receptors, which are receptors for oxidized LDL, such as dextran sulphate, polyinosinic acid, heparin and acetylated LDL also significantly induced M-CSF production from human monocytes, although this was at levels below 2 ng/ml. These results indicate that serum modified by oxidation stimulates monocytes to produce M-CSF resulting in their proliferation, and that signalling via scavenger receptors is one of the mechanisms responsible for this induction of M-CSF.

Intravenously administered macrophage colony-stimulating factor (M-CSF) specifically acts on the spleen, resulting in the increasing and activating spleen macrophages for cytokine production in mice.

IL-6 was transiently expressed in sera of mice after a bolus intravenous injection with LPS and it peaked 2 h later. Intravenous administration of M-CSF at 250 micrograms/kg/day for 5 days prior to an injection of 25 micrograms/kg of LPS elevated the serum IL-6 level 10-fold higher than that of mice which were not given M-CSF. Although M-CSF had no effect on the number of macrophages in alveoli and peritoneal cavity, it tripled the number of spleen macrophages and increased macrophage-progenitor cells 7-fold when injected intravenously. Spleen macrophages from M-CSF-injected mice produced 5-fold more IL-6 in response to LPS-stimulation in-vitro. However, M-CSF-injection had lesser effects on LPS-induced IL-6 production from liver, alveolar and peritoneal macrophages. Exogenously administered M-CSF was detected at higher concentration and for longer duration in the spleen than in any other organs examined. Spleen macrophages incubated in-vitro with more than 1000 U/ml of M-CSF for 3 days also produced more LPS-induced IL-6 than untreated cells. These results indicate that intravenously administered M-CSF not only enhances macrophage development in the spleen, but also primes mature macrophages for cytokine production.

Macrophage colony-stimulating factor (M-CSF) augments cytokine induction by lipopolysaccharide (LPS)-stimulation and by bacterial infections in mice.

We studied the effects of M-CSF on cytokine induction in vivo by LPS or by bacterial infection by comparing between the serum cytokine levels of mice administered with and without M-CSF. M-CSF at 250 micrograms/kg/day for 3 days significantly augmented serum IL-6 level induced by a subsequent injection of 25 micrograms/kg of LPS. The augmented IL-6-induction was dose-dependent from 50 to 1250 micrograms/kg/day of M-CSF, and required 2- to 3-doses of M-CSF at 250 micrograms/kg/day. Mice primed with M-CSF induced IL-6 in response to a 5-fold lower dose of LPS, and also produced higher levels of IL-1 alpha, IL-10, GM-CSF, TNF-alpha, and IFN-gamma than control mice. The priming effect of M-CSF was transient and reversible, and elicited independently of T-cells. An injection with intact bacteria, such as Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus also induced IL-6 in normal mice, and M-CSF administration augmented the induction of these cytokines. These results showed that M-CSF positively regulates LPS-dependent and -independent cytokine induction, suggesting a defensive effect against infectious agents through enhanced cytokine production.

Effects of macrophage colony-stimulating factor (M-CSF) on lipopolysaccharide (LPS)-induced mediator production from monocytes in vitro.

M-CSF is a macrophage-lineage-specific growth factor that causes proliferation and differentiation of progenitor cells in the bone marrow. To investigate the effects of M-CSF on more matured cells, human monocytes were cultured in the presence or absence of M-CSF for 6 days. Addition of M-CSF at more than 10(2) U/ml resulted in higher viability and caused morphological differentiation to large macrophage-like cells. LPS-induced mediator production was also compared between M-CSF-treated and control cell. Monocytes were incubated with or without M-CSF for 3 days, and were stimulated with 1 microgram/ml of LPS for 2 days. IL-1 beta was not detected in the both culture supernatants, and PGE2 production was not influenced by M-CSF. However, amounts of G-CSF, GM-CSF, IL-6, and TNF-alpha produced in response to 1 microgram/ml of LPS were 1.5 to 2 times greater from monocytes treated with 10(4) U/ml of M-CSF than from control cells. The priming effect of M-CSF on LPS-induced cytokine production was found to require 3-day preincubation, and reached a maximum at the concentration of 10(4) U/ml. M-CSF-treated cells responded to a 10 times lower concentration of LPS than control cells in terms of cytokine production. M-CSF was also shown by flowcytometric analysis to influence the expression of CD14, a receptor for LPS, which might render monocytes more sensitive to LPS.

Effect of recombinant human macrophage-colony stimulating factor on marrow, splenic, and peripheral hematopoietic progenitor cells in mice.

The effects of human macrophage colony-stimulating factor (M-CSF) on marrow, splenic, and peripheral progenitor cells (CFU-M, CFU-GM, and CFU-G) were investigated in mice administered recombinant human M-CSF (8-4,000 micrograms/kg). Single injection of 4,000 micrograms/kg of M-CSF resulted in a decrease in the number of marrow progenitor cells (CFU-M, CFU-GM, and CFU-G) on day 2 followed by a gradual increase, returning to the original level on day 4 or 5. In contrast, each type of splenic progenitors tested for started to increase markedly on day 2, reaching a level 4- to 15-fold higher than that of the basal value on day 3 or 4. Peripheral CFU-M, CFU-GM, and CFU-G also increased on day 2. In addition, administration of 800 micrograms/kg of M-CSF in mice caused a decrease in marrow CFU-G, as well as an increase in splenic CFU-G. The present results indicate that treatments of mice with pharmacological concentrations of human M-CSF affect the number of progenitor cells not only of monocyte/macrophage lineage but also of granulocyte lineage. Also, the coincidence between decrease of marrow progenitor cells and increase of splenic and peripheral progenitor cells suggests that the progenitor cells are released from bone marrow to peripheral blood and reseeded to the spleen by the action of M-CSF.

Production of human antithrombin-III in a serum-free culture of CHO cells.

A simple method was developed to establish serum-independent Chinese hamster ovary (CHO) cells that grew and secreted high level of human antithrombin-III (AT-III). First, human AT-III and mouse dihydrofolate reductase (DHFR) cDNAs were transfected into DHFR-deficient CHO cells. Transfected cells were treated with increasing concentrations of methotrexate (MTX) and clones secreting high levels of AT-III (10-20 micrograms/ml/3 day) in a serum-containing medium were obtained. Serum-independent clones were derived from the serum-dependent clones by simply culturing the cells for a few weeks in a serum-free medium. In a serum-free medium the established serum-independent clones grew at normal rate and produced almost equivalent amount of AT-III to that of the serum-dependent, parent clones. In addition, AT-III from the serum-independent clones has specific activity similar to that of plasma-derived AT-III.