Effect of prolactin on the level of corticosterone in the blood and synthesis of lymphocyte-activating factors by macrophages under conditions of glucocorticoid loading

The present study examines in vivo the interaction between glucocorticoids and prolactin, and immunomodulating activity of prolactin, including prolactin-induced changes in production of lymphocyte-activating factors and alteration in humoral immune response. It was shown that prolactin application increased the level of corticosterone in the rat blood. Administration of prolactin prior to hydrocortisone administration induced alteration in the level of corticosterone that depended on the dose of hydrocortisone. Application of hydrocortisone reduced humoral immune response and lymphocyte-activating factors production by peritoneal macrophages. Administration of prolactin prior to hydrocortisone administration prevents inhibitory action of glucocorticoids/on humoral immune response and lymphocyte-activating factors production by macrophages.     

Interference of polyester inflammation on the development of Yoshida ascites hepatoma in the rat

Minced polyester threads introduced into the peritoneal cavity cause a chronic inflammation with evidence of macrophage and lymphocyte stimulation. In this paper an interference between this kind of inflammation and the growth of Yoshida ascites hepatoma has been shown, which has been found to dependent on the time interval elapsed between the introduction of polyester (Mersilene) minces and injection of the hepatoma cells. Rats were treated intraperitoneally with Mersilene and then divided in to three groups: the first was injected intraperitoneally with hepatoma cells immediately (TM 0), the second after 7 (TM 7) and the third after 14 days (TM 14); rats untreated with polyester and implanted with the same number of hepatoma cells served as controls (NT). While in groups NT and TM 0 a rapid growth of hepatoma cells occurred, together with the accumulation of a considerable volume of ascitic fluid, no tumor growth neither ascite production occurred in groups TM 7 and TM 14; in these animals where several days were allowed to elapse after polyester introduction, the hepatoma cells which had been injected rapidly disappeared and were no more found 48 h after the intraperitoneal injection. It is suggested that the inhibition of the neoplastic growth may be dependent on the activation of macrophages (and possibly NK cells) which accompanies the development of the chronic polyester inflammation.     

Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages.

The effect of glucocorticoids on the production of NO2- and NO by the macrophage cell line J774 was investigated. Stimulation of the cells with lipopolysaccharide (LPS) resulted in a time-dependent accumulation of NO2- in the medium, reaching a plateau after 48h. Concomitant incubation of the cells for 24h with dexamethasone (0.001-1.0 microM) or hydrocortisone (0.01-10.0 microM) caused a concentration-dependent inhibition of NO2- formation. The cytosol of J774 cells stimulated with LPS and IFN-gamma produced a time-dependent increase in the release of NO. This was blocked in a concentration-dependent manner by dexamethasone and hydrocortisone, but not progesterone, administered concomitantly with the immunological stimulus. None of these compounds had any effect on the release of NO once the enzyme had been induced. The inhibitory effect of hydrocortisone on NO formation was blocked by cortexolone. These data suggest that part of the anti-inflammatory and immunosuppressive actions of glucocorticoids is due to their inhibition of the induction of the NO synthase.     

Inhibition of proliferation of normal and neoplastic liver cells under conditions of prolonged hormonal stimulation

Single injections of rats with hydrocortisone led to the inhibition of regenerating liver cell proliferation and protooncogene++ Ha-ras mRNA synthesis within 48 hours of hormonal induction. Administration of hydrocortisone to rats daily for 10 days resulted in a persistent decrease of the liver cell capacity to proliferate in response to partial hepatectomy. This inhibiting effect was observed for at least 7 days after cessation of hormonal stimulation; the level of Ha-ras mRNA was thereby decreased. A marked inhibition of ascite hepatoma cell growth was demonstrated after injections of those cells to mice induced with hydrocortisone for 10 days. Such a persistent effect of hydrocortisone is thought to be due to the depletion of the hormone-dependent hepatotrophic factors. The effect of the glucocorticoid hormone in vivo can be supposed to involve both the direct and indirect regulation of target cell proliferation. The latter is mediated via the changes in the activity of exogenous factors which control cell growth and proliferation.     

Immunological reactivity of the lung: VII. Effect of corticosteroids and cyclophosphamide on the Fc receptor function of alveolar macrophages

The effects of various in vitro and in vivo regimens of either corticosteroid or cyclophosphamide administration on guinea pig alveolar macrophages were studied. Corticosteroid- and cyclophosphamide-induced immunosuppression was assessed by the effect of drug administration on the capacity of alveolar macrophages to attach to and/or ingest antibody-coated sheep red blood cells (SRBC). In vitro hydrocortisone (up to 20 μg/ml) had no effect on either the binding or ingestion of antibody-coated SRBC. Two separate regimens of in vivo corticosteroids were given: a single dose of iv hydrocortisone (100 mg/kg), which is a short-acting soluble preparation, and sc doses of cortisone acetate (100 mg/kg for 7 days), which is a depot preparation resulting in sustained levels of plasma cortisol of the magnitude of that found for a brief period of time following iv injection of hydrocortisone. Both regimens resulted in similar degrees of peripheral blood lymphocytopenia and monocytopenia 4 and 24 hr, respectively, following injection. The regimen of hydrocortisone has previously been reported to have no effect on alveolar macrophage cytotoxic effector function in antibody-dependent cellular cytotoxicity (ADCC), whereas the cortisone acetate regimen markedly suppressed ADCC. In the present study, hydrocortisone had no effect on either the binding or ingestion of antibody-coated SRBC by alveolar macrophages. In contrast, cortisone acetate caused a marked decrease in both the binding and ingestion of antibody-coated SRBC. This suppressive effect was maximal at suboptimal concentrations of antibody on the SRBC and could be overcome by increasing the concentrations of anti-SRBC antibody. Alveolar macrophages from animals treated with daily cyclophosphamide (a regimen which suppresses ADCC) were capable of binding and ingesting antibody-coated SRBC normally. Thus, prolonged exposure to corticosteroids in vivo causes an alteration in membrane Fc receptor function of alveolar macrophages, which can explain this impaired ability to kill target cells. Since cyclophosphamide therapy did not interfere with the binding and ingestion of antibody-coated target cells, it is concluded that the impairment in killing of target cells by alveolar macrophages is not directly related to an alteration of Fc receptor function but to a defect in the actual killing process.     

P cell stimulating factor and glucocorticoids oppose the action of interferon-gamma in inducing Ia antigens on T-dependent mast cells (P cells)

The expression of Ia antigens of cultured lines of T-dependent mast cells (TDMC) or persisting (P) cells was modulated by three hormones: interferon-gamma (IFN-gamma), P cell-stimulating factor (PSF), and glucocorticoids. The induction of Ia antigens by cloned IFN-gamma was inhibited by a homogeneous preparation of PSF, the T cell-derived factor that TDMC require for their in vitro growth. This inhibitory effect of PSF was dose-dependent and could not be overcome by increasing the levels of IFN-gamma. Other cytokines such as granulocyte/macrophage colony-stimulating factor, T cell growth factor, and L cell-derived macrophage colony-stimulating factor had no inhibitory effect. Thus, two different T cell lymphokines, PSF and IFN-gamma, have opposing effects on the expression of Ia antigens on TDMC. Glucocorticoids (hydrocortisone, corticosterone, dexamethasone, prednisolone, and fludrocortisone) antagonized the induction of Ia antigens by IFN-gamma, whereas sex steroids (progesterone, estradiol, and testosterone) and a mineralocorticoid (aldosterone) did not. The inhibitory effect of glucocorticoids could not be overcome by increasing concentrations of IFN-gamma, but was significantly inhibited by progesterone (10(-6) M), indicating the likely involvement of typical glucocorticoid receptors. In contrast to their effectiveness on macrophages, prostaglandin E2 and dibutyryl cyclic AMP only slightly inhibited the induction of Ia antigens on TDMC by IFN-gamma, whereas endotoxin (1 to 60 micrograms/ml) had no effect.     

Glucocorticoids inhibit formation of inositol phosphates in macrophages.

Glucocorticoids inhibited the zymosan-induced formation of inositol phosphates in macrophages. No inhibition was observed with progesterone. Inhibitors of protein (cycloheximide) and RNA (actinomycin D) synthesis exhibited similar inhibitory effects. The activity of phospholipase C in subcellular fractions was not altered by hormone treatment of the cells. However, the incorporation of inositol into membrane lipids was reduced by dexamethasone. These data indicate that glucocorticoids are able to inhibit the formation of inositol phosphates; the effect of the hormone is rather due to an inhibition of the incorporation of inositol in membrane lipids than to an inhibition of phospholipase C. The anti-inflammatory action of glucocorticoids may, therefore, also be attributed to their effect on the polyphosphoinositide cycle and inositol phosphate-mediated processes.     

Suppressive effect of streptomycin on the phagocytic activity of mouse peritoneal macrophages for Histoplasma capsulatum

Streptomycin inhibited the phagocytic activity of mouse peritoneal macrophages forHistoplasma capsulatum. The inhibitory effect was demonstrable following both in vitro and in vivo administration of drug. The observations from examination by direct smear were confirmed by culturing for viable phagocytized organisms. A simple and reproducible technique for the counting of viable phagocytized organisms was developed. Forty-eight hours in vitro treatment of macrophage cultures with 10 to 200 µg/ml of streptomycin produced a graded inhibition of phagocytic activity, minimal at 10 µg/ml and maximal at 200 µg/ml of streptomycin. The parenteral administration of streptomycin significantly reduced phagocytic activity of mouse peritoneal macrophages forH. capsulatum. Mice were treated daily with the subcutaneous injections of 5, 2.5 or 1 mg streptomycin or saline. At 7, 14, 21 and 28 days post-treatment phagocytic activity of macrophages obtained from these mice was tested. There was a progressive, dose-dependent decrease in the phagocytic activity of macrophages derived from streptomycin-treated mice.     

Growth regulation of transformed T cells by nonactivated macrophages: the role of Ia expression

We studied the influence of unactivated mouse peritoneal macrophages on the proliferative capacity of a spontaneously transformed MRL-lpr/lpr T cell clone. Macrophages, 25%, induced a reduction in proliferative rate from 20% to 95% measured by [3H]thymidine incorporation and microscopic cytometry. MHC-compatible (H-2k) macrophages caused growth inhibition reciprocal to the amount of Ia expression on the macrophage. Thus, with increasing preculture of the macrophages there was both decreasing Ia and increasing suppression. H-2-incompatible macrophages had maximal inhibitory capacity without preincubation. Macrophages derived from the peritoneum of MRL-lpr/lpr mice were less suppressive than macrophages from other H-2k mice. In contrast to the case of activated macrophages in other studies, in the present system there was no killing of T cells, only reduction in proliferation. The inhibitory effect of the macrophages correlated with the spontaneous formation of rosettes between the macrophages and the T cell clone. The number of rosettes forming a single layer of T cells around the macrophages, but not the number of rosettes with multiple layers of cells, was reciprocally related to the amount of Ia expression. The results suggest that macrophages bear a surface structure that influences and modulates the growth of T cells.     

Macrophage activation by tuftsin and muramyl-dipeptide

Summary Peritoneal macrophages from tuftsin or MDP-treated mice were tested for their cytostatic activity for tumor cell proliferation. Both substances are able to activate macrophages either after intravenous injection or after incubation in vitro with normal macrophages. But a stimulation as well as an inhibition of tumor cell growth can result from macrophage activation depending on the timing and dose injected. Restoration of the impaired cytostatic capacity of macrophages of mice observed with aging, is obtained by repeated administration of tuftsin. Normal and BCG-stimulated macrophages were examined for their regulatory activity on the proliferation of P815 tumor cells. Low density of macrophages per well determines a stimulation of target cell growth whether the macrophages are normal or activated. When the number of macrophages is increased, under conditions in which normal macrophages are not inhibitory. BCG-stimulated macrophages exert already a strong cytostatic activity. At high macrophage content it appears that normal macrophages can also display an inhibitory activity. Macrophage-tumor cell interactions are highly dependent on the concentration and the state of activation of macrophages.Reprint requests should be addressed to M. Bruley-Rosset     

Macrophage-derived neutrophil chemotactic factor is involved in the neutrophil recruitment inhibitory activity present in the supernatants of LPS-stimulated macrophages

In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-alpha and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal(-)) and bound (D-gal(+)) fractions, with MNCF being found in the D-gal(+) fraction. Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal(-) fraction. Furthermore, the incubation of the D-gal(-) fraction with anti-TNF-alpha plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal(+) activity. Overall, these results suggest that the D-gal(-) inhibitory effect is partially mediated by TNF-alpha and IL-8, and that MNCF accounts for the inhibition of neutrophil migration in vivo by the D-gal(+) fraction.     

Carnosine as a stimulator of cytotoxic and phagocytic function of peritoneal macrophages]

Biochemical changes in peritoneal macrophages and their relatedness to the cytostatic and phagocytotic function in C3HA mice injected with a single intraperitoneal dose of 0.45 mM carnosine and 4-methyluracil or stimulated with peptone have been studied. During the first 24 hours after injection both carnosine and 4-methyluracil increase the activity of adenosine deaminase and purine nucleoside phosphorylase, the key enzymes of purine catabolism which is the main source of O2-. radicals in macrophages. In carnosine-stimulated macrophages the activity of membrane 5'-AMP nucleotidase decreases on days 1-3 after injection which points to alleviation of adenosine-induced inhibition as well as to macrophage activation. Carnosine increases the cytostatic and phagocytotic activities of macrophage coupled to O2-. production. The mechanism of the stimulating effect of carnosine on macrophages seems to consist in the dipeptide interaction with specific receptors localized on the plasma membrane of macrophagal cells.     

Suppressive effect of delta 9-tetrahydrocannabinol in vitro on phagocytosis by murine macrophages

Incubation of normal mouse peritoneal cells consisting of over 90% phagocytizing macrophages with delta 9-tetrahydrocannabinol (THC) resulted in a inhibition of phagocytic function. The THC in a dose-related manner suppressed the percentage of macrophages per culture which ingested yeast and the average number of yeast particles ingested by the phagocytizing macrophages. The vehicle used to suspend the THC in vitro, i.e., DMSO, had no detectable effect on macrophage function. Suppression of phagocytosis with no effects on viability or cell number occurred with doses of 10 micrograms or less THC per milliliter culture medium. Measurable suppression also occurred after 24- to 48-hr treatment of the macrophages with the THC. This compound had little if any detectable effect on phagocytosis when added directly to the cultures shortly before testing for phagocytosis. Further studies concerning the effects of THC on macrophage function appear warranted.     

Regulation of the phospholipid turnover rate and protein kinase C activity as a necessary stage in the realization of the growth-inhibiting effect of dexamethasone on hepatoma 22 cells]

The role of protein kinase C and phospholipid turnover in the realization of the cytostatic effect of dexamethasone on hormone-sensitive cells of mouse hepatoma 22 has been studied. It was found that dexamethasone added to hepatoma cells induces a rapid (within 30 min) inhibition of the protein kinase C activity with a simultaneous decrease of the 32P incorporation into the major phospholipids (phosphatidylglycerol, phosphatidylcholine, and phosphoinositides). Analysis of correlation between the protein kinase C activity and phospholipid turnover rate revealed that phosphatidylglycerol and phosphatidylcholine synthesis is under the positive control of protein kinase C, whereas that of phosphoinositides is not controlled by the enzyme. A proportional decrease in the rates of metabolism of all the three major phospholipids after addition of the hormone to hepatoma cells suggests that inhibition of phospholipid turnover is one of the primary manifestations of the dexamethasone effect. The hormone-induced decrease in the protein kinase C activity may be regarded as being due to these changes.     

Action of hydrocortisone on the cytotoxic reactions in delayed hypersensitivity to microbial antigens

A study was made of the effect of hydrocortisone (HC) injected to animals with delayed hypersensitivity (DH) to BCG antigens on the cytotoxic activity of lymphocytes and production of lympho- and macrophage toxins. The cytotoxic test with the use of sensitized lymphocytes and preparation of lympho- and macrophage toxins were performed in vitro in the presence of specific microbial antigens. It was shown that HC exerts the most intense inhibitory action on the production of macrophage toxin. High doses of the hormone also inhibited the production of lymphotoxin. At the same time the cytotoxic activity of lymphocytes of the lymph nodes in DH was not inhibited by the employed doses of HC. No reduction was seen either in the sensitivity of autologous adhesive cells (macrophages) used as target cells for studying the cytotoxic activity of lymphocytes.     

Changes in the activity of the brain renin-angiotensin system and in the functional state of the pituitary-adrenal system of rats under the influence of captopril]

While studying the effect of peroral captopril injections on the activity of renin-angiotensin system (RAS), the activity of angiotensin-converting enzyme (ACE) and angiotensin II content from anterior and mediobasal hypothalamus, medulla and adenohypophysis of intact rats has been established to decrease. Captopril administration decreases ACE activity which increases after hydrocortisone injection in rat medulla and striatum. Captopril results in no potentiation of hormonal effect in hypothalamus and in adenohypophysis where ACE activity decreases following hydrocortisone injection. A decrease in the RAS activity of brain structures and adenohypophysis induced by captopril administration to rats is accompanied by the inhibition of the activity in the pituitary-adrenal system. A decrease in ACTH level and in 11-hydroxycorticosteroids of the rat blood plasma has been determined after single captopril injection in the dosage of 10 and 50 mg/kg of body weight. Duration of the effect depends on the captopril dosage.     

Further studies on the inhibition of chemical carcinogenesis by Corynebacterium parvum

Summary The conditions under which administration of Corynebacterium parvum (CP) inhibits the development of tumours in response to methylcholanthrene (MC) have been further delineated. After subcutaneous (SC) injection of a single large dose of MC in solution (0.5 mg to CBA mice, 0.25 mg to BALB/c), repeated intravenous (IV) injection of CP starting 4 days before injection of the carcinogen delays or prevents the development of fibrosarcomas, whereas a similar course of injections starting 4 weeks later, and single injections of CP at various times, have little or no inhibitory effect. After injection of only 0.1 mg MC repeated injection of CP, even when started before injection of carcinogen, is ineffective. Carcinogenesis may also be inhibited by repeated IV injection of CP starting 4 days before implantation of Millipore discs impregnated either with MC alone or with a mixture of MC and paraffin wax. In untreated mice SC implantation of a disc impregnated with 0.1 mg MC was more strongly carcinogenic than SC injection of the same quantity of MC in solution. The effect of CP depended on the site of implantation, the type of disc and the regimen of CP administration. With MC wax discs implanted in the peritoneal cavity the development of tumours was completely prevented by repeated administration of CP starting before insertion of the disc. Similar treatment resulted in more modest inhibition of carcinogenesis after SC implantation of MC wax discs and MC discs of small pore size. In previous papers three possible mechanisms have been suggested to account for the inhibitory effect of CP on chemical carcinogenesis by MC: (1) destruction of transformed cells by CP-activated macrophages; (2) accelerated conversion of the MC to completely non-carcinogenic substances; and (3) inhibition of the conversion of MC to a highly carcinogenic epoxide. The present results do not exclude any of these possibilities but the observation that single injections of CP have little effect suggests that modification of the metabolism of MC is not the whole explanation. The different results obtained with carcinogenic discs implanted in different sites, and with different types of disc, are attributed to differences in the degree of macrophage activation and in the extent to which activated macrophages made effective contact with the carcinogen and with developing tumour cells. Two possible explanations of the failure of repeated injection of CP to reduce the incidence or rate of development of tumours in response to injection of only 0.1 mg MC are suggested.     

Rat macrophage activation after treatment with the bleomycin group of antitumour antibiotics in vivo

Summary We have previously reported that bleomycin and its derivative peplomycin enhance the release of cytokines by rat spleen cells during mitogen-stimulated cell culture in vitro, but liblomycin, another derivative of bleomycin, decreases cytokine release to below untreated control levels. Cytokine release correlated well with the inhibition of subcutaneous tumour growth after treatment with equivalent doses of the three analogues. In contrast, ascites tumour growth is completely inhibited by liblomycin and appears to be at least partly macrophage-mediated because the antitumour effect can be significantly inhibited by carageenan. This study shows that bleomycin and its analogues activate rat peritoneal macrophages and increase interleukin-6 release, O₂ ^– production, cell spreading, phagocytosis and random migration of macrophages, but only bleomycin enhances peritoneal macrophage invasion into a monolayer of rat lung endothelial cells in vitro. This study also shows that although liblomycin decreases spleen cell cytokine production and is less effective than bleomycin against subcutaneous tumour, as we have previously reported, the antitumour drug activates peritoneal macrophages and, compared to bleomycin, has a remarkable therapeutic effect on rat ascites tumour.     

Effect of a chalone-containing preparation from Ehrlich ascites tumor on DNA synthesis and cell division in the tumor in relation to the time of day

The mitosis inhibitory action of chalone-containing preparation of the Ehrlich ascite tumour was shown to depend on the time of its administration on round the clock, and on the circadian rhythm phase of the mitotic activity in this tumour. This allowed a conclusion that the chalone system of the tumour may be involved in the formation of the circadian rhythm of cell division. It was found that Ehrlich's ascite tumour chalone system regulated DNA synthesis influencing the cell passage from G1-phase of the mitotic cycle to S-phase, and the processes occurring during S-phase.     

Oxidized lipoproteins suppress nitric oxide synthase in macrophages: study of glucocorticoid receptor involvement

Activated cholesterol-laden macrophages in atherosclerotic lesions are believed to influence the progression of this disease. The induction of nitric oxide synthase (iNOS) activity was investigated in control and cholesterol-laden J774 macrophages, obtained by pre-incubation with oxidized or acetylated low density lipoproteins (oxLDL, acLDL). Loading with oxLDL caused a small induction of NOS activity in unstimulated cells, as indicated by nitrite and citrulline accumulation in the supernatant. However, it suppressed the iNOS activity resulting from stimulation of the cells with lipopolysaccharide with or without interferon-gamma. AcLDL had no inhibitory effect, indicating that cholesterol accumulation as such was not responsible. Since the induction of NOS in macrophages is inhibited by glucocorticoids, the possibility that a glucocorticoid-like factor, formed during oxidation of LDL, may cause the inhibition, was investigated. However, addition of the glucocorticoid receptor antagonist mifepristone did not prevent the oxLDL-dependent NOS inhibition, indicating that the glucocorticoid receptor is not involved in the suppressive effect of oxLDL.