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Patent US4822776 - Lipoprotein lipase suppression by endotoxin-induced mediator (shock assay) - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign in<nobr>Advanced Patent Search</nobr>PatentsA mediator substance exhibiting inhibitory effect upon anabolic enzyme activity in mammals, is prepared by a method comprising gathering a sample of macrophage cells from a mammal, incubating a portion of the macrophage cells with a stimulating mate This invention was made in the course of a grant from...http://www.google.com/patents/US4822776?utm_source=gb-gplus-sharePatent US4822776 - Lipoprotein lipase suppression by endotoxin-induced mediator (shock assay)Advanced Patent SearchPublication numberUS4822776 APublication typeGrantApplication numberUS 06/792,372Publication dateApr 18, 1989Filing dateOct 29, 1985Priority dateSep 8, 1981Fee statusLapsedPublication number06792372, 792372, US 4822776 A, US 4822776A, US-A-4822776, US4822776 A, US4822776AInventorsAnthony Cerami, Masanobu KawakamiOriginal AssigneeThe Rockefeller UniversityExport CitationBiBTeX, EndNote, RefManPatent Citations (2), Non-Patent Citations (157), Referenced by (62), Classifications (45), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetLipoprotein lipase suppression by endotoxin-induced mediator (shock assay)US 4822776 AAbstract A mediator substance exhibiting inhibitory effect upon anabolic enzyme activity in mammals, is prepared by a method comprising gathering a sample of macrophage cells from a mammal, incubating a portion of the macrophage cells with a stimulating mate
We claim: 1. A pharmaceutical composition for the treatment of obesity in humans, comprising:A. a proteinaceous mediator substance capable of suppressing the activity of the anabolic enzymes lipoprotein lipase, acetyl Coenzyme A carboxylase and fatty acid synthetase, and inhibiting the growth and differentiation of erythroid-committed cells, or a specific binding partner thereto, wherein said mediator substance exhibits molecular weight levels of about 300,000, 70,000 and in the range of 400 to 1000 Daltons as determined by gel electrophoresis, and is derived from animal macrophage cells that have been incubated with a stimulator material that accompanies an invasive stimulus; and B. a pharmaceutically effective carrier. 2. The composition of claim 1 wherein said mediator substance is further capable of inhibiting the differentiation of fat cells, and increasing the activity of hormone-sensitive lipase in fat cells and the uptake of glucose in muscle cells, while demonstrably lacking leucocyte activator activity and the ability to cause either fever or muscle protein degradation.
RELATED APPLICATIONS The instant application is a division of Ser. No. 414,098, filed Sept. 7, 1982, which is in turn continuation-in-part of Ser. No. 351,290, now abandoned, filed Feb. 22, 1982, by the same applicants, which in turn is a continuation-in-part of Ser. No. 299,932, now abandoned, filed Sept. 8, 1981. Applicants claim the benefit of these applications under 35 U.S.C. �120.
RELATED PUBLICATIONS The applicants are authors or coauthors of two articles directed to the subject matter of the instant invention: (1) [applicants only] "Studies of Endotoxin-Induced Decrease in Lipoprotein Lipase Activity", J. EXP. MED. 154 at 631-639 (September, 1981, published after Sept. 8, 1981), incorporated herein by reference; and (2) [co-authors with Phillip H. Pekala and M. Daniel Lane]: "Lipoprotein Lipase Suppression in 3T3-L1 Cells by an Endotoxin-Induced Mediator from Exudate Cells", PROC. NAT'L. ACAD. SCI. 79 at 912-916 (February, 1982, published after Feb. 22, 1982), also incorporated herein by reference.
Several common physiological and biochemical derangements have been seen in various mammalian hosts responding to variety of invasive stimuli such as bacterial, viral and protozoan infections, as well as tumors and endotoxemia. For example, these responses include fever, leukocytosis, hyperlipidemia, reduced food intake and activity, and other modifications in muscle, white blood cell and liver metabolism. Recently, a hypertriglyceridemia in rabbits infected with a protozoan parasite, Trypanosoma brucei was reported by C. A. Rouser and A. Cerami, MOL. BIOCHEM. PARASITOL. 1 at 31-38 (1980). The reported hypertriglyceridemia was accompanied by a marked decrease in the activity of the enzyme lipoprotein lipase (LPL) in peripheral tissues.
LPL activity has been observed by others, and it has been noted that this condition has existed when the human body was in shock. See E. B. Man, et al, "The Lipids of Serum and Liver in Patients with Hepatic Diseases", J. CLIN. INVEST. 24 at 623, et seq. (1945); See also John I. Gallin, et al, "Serum Lipids in Infection", N. ENGL. J. MED. 281 at 1081-1086 (Nov. 13, 1969); D. Farstchi, et al., "Effects of Three Bacterial Infections on Serum Lipids of Rabbits", J. BACTERIOL. 95 at 1615, et seq. (1968); S. E. Grossberg, et al., "Hyperlipaemia Following Viral Infection in the Chicken Embryo: A New Syndrome", NATURE (London) 208 at 954, et seq. (1965); Robert L. Hersch, et al., "Hyperlipidemia, Fatty Liver and Bromsulfophthalein Retention in Rabbits Injected Intravaneously with Bacterial Endotoxin", J. LIPID. RES. 5 at 563-568 (1964); and Osamu Sakaguchi, et al., "Alterations of Lipid Metabolism in Mice Injected with Endotoxins", MICROBIOL. IMMUNOL. 23 (2) at 71-85 (1979); R. F. Kampschmidt, "The Activity of Partially Purified Leukocytic Endogeneous Mediator in Endotoxin-Resistant C3H/HeJ Mice", J. LAB. CLIN. MED. 95 at 616, et seq. (1980); and Ralph F. Kampschmidt, "Leukocytic Endogeneous Mediator", J. RET. SOC. 23 (4 ) at 287-297 (1978).
SUMMARY OF THE INVENTION In accordance with a first aspect of the present invention, a method for preparing a mediator substance for use in assessing the state of anabolic enzymes in mammals, is disclosed, which finds particular utility in the instance where the mammals are undergoing invasive stimmuli such as, viral agents, bacteria, protozoa, tumors, endotoxemia and others. In its simplest aspect, the method comprises gathering a sample of macrophage cells from a mammal and incubating a portion of the macrophage cells with a stimulator material associated with an invasive event. For example, the stimulator material may be endotoxin, in the instance of endotoxemia, trypanosomes, in the instance of the above mentioned protozoan parasite Trypanosoma brucei, and others.
The invention includes methods for detecting the presence of samples of the various invasive stimuli in mammals by measuring mediator substance activity in the mammals. Thus, a number of mediator substance may be prepared from the incubation of individual cell samples with known stimulator materials, and these mediator samples may thereafter be used to raise antibodies capable of specifically detecting the presence of the respective mediator substances. These antibodies may be prepared by known techniques, including the well known hybridoma technique for example, with fused mouse spleen lymphocytes and myeloma, or by development in various animals such as rabbits, goats and other mammals. The known mediator samples and their antibodies may be appropriately labelled and utilized to test for the presence of the mediator substance in, for example, serum, as one may measure the degree of infection, and determine whether infection is increasing or abating, by observing the activity of the mediator substance therein. A variety of well known immunological techniques may be utilized in accordance with this aspect of the present invention, including single and double antibody techniques, utilizing detectible labels associated with either the known mediator substances, or their respective associated antibodies.
The preparation of the mediator substance, and the determination of the importance of its activity, has resulted in the development of numerous avenues of diagnostic and therapeutic application. It is clear from the foregoing and following, that the detection of invasive stimuli may be made by the identification of the mediator substance, either directly or through the development of antibodies useful in immunological diagnosis. Further, these same antibodies may be utilized for direct treatment by control of mediator activity, to avert the development of shock in mammals, while the mediator substance may be utilized as screening agents in an assay system for the identification of drugs, agents and other substance capable of neutralizing the adverse effects of the mediator substance, and thereby providing treatment of the adverse sequelae of infection.
It is yet further object of the present invention to provide a method for the treatment of mammals to control the activity of said mediator substance so as to mitigate or avert the adverse consequences of their activity.
DESCRIPTION OF THE DRAWINGS FIG. 1A shows the effect of serum from endotoxin-sensitive mice treated with endotoxin on adipose tissue LPL activity in endotoxin-sensitive mice. Mediator activity was observed and conclusions drawn as set forth in Example 1, paragraph E herein. The data are expressed as the mean (�SEM) of six animals for each group.
FIG. 1B shows the effect of serum from endotoxin-sensitive mice treated with endotoxin on adipose tissue LPL activity in endotoxin-resistant mice. The data are expressed as the mean (�SEM) of three animals for each group.
FIG. 2 shows the effect of medium from exudate cell cultures on adipose tissue LPL in endotoxin-resistant mice. The data are presented as the mean (�SEM) of four or five animals.
FIG. 3 shows the effect of conditioned medium from endotoxin-treated mouse peritoneal exudate cells over lipoprotein lipase activity of 3T3-L1 cells. Data are expressed as mean SEM (n=4).
FIG. 4 shows the effect of conditioned medium from endotoxin-treated mouse peritoneal exudate cells on the activities of acetyl CoA carboxylase and fatty acid synthetase in 3T3-L1 cells. Three hundred (300) μl of conditioned medium was added to cultures of 3T3-L1 cells (4.2�106 cells/dish) in 6 cm dishes containing 3.5 ml of DME medium and 10% fetal calf serum. After the indicated times of incubation, the enzymatic activity of acetyl CoA carboxylase (identified by the symbol " ") and fatty acid synthetase (identified by the symbol "o") on a digitonin releaseable cytosolic fraction of the cells was assessed.
FIG. 5 shows the effect of mediator that suppresses the synthesis of acetyl CoA carboxylase. At the indicated times after exposure of the 3T3-L1 cells to the mediator (300 μl of conditioned medium, the cells were pulse-labeled with 0.5 mCi of 35 S-methionine for 1 hour. Cytosolic fractions were obtained by digitonin treatment of a monolayer. Aliquots of the cytosolic fractions (2�105 cpm for all determinations) were incubated with anti-acetyl CoA carboxylase and the immunoprecipitable material isolated and characterized as described in Example II, infra. Panel A: Autoradiogram of a 7.5%-acrylamide-0.1% SDS gel analysis of immunoadsorbable protein. Lane 1--control, without exposure to mediator; Lanes 2, 3, and 4--exposure of the cells to the mediator for 3, 6 and 20 hours, respectively. Panel B: Results of a densitometric scan of the autoradiogram, indicating percent of immunoadsorbable material remaining relative to control, after exposure to the mediator.
FIG. 7 shows the effect of the mediator on 35 S-methionine incorporation into protein. 3T3-L1 cells were incubated with 300 μl of conditioned medium from endotoxin-treated mouse peritoneal exudate cells for the appropriate period and protein pulse-labeled with 0.5 mCi of 35 S-methionine for 1 hour. Soluble proteins were obtained by digitonin treatment of the cells, the remainder of the monolayer was extracted with NP-40 and a membrane protein fraction obtained. Incorporation of 35 S-methionine into acid precipitable material was determined as described in Example II, infra. The incorporation of radioactivity into soluble protein () or membrane protein () following exposure of the cells to the mediator are shown for the indicated time.
FIG. 8 shows the effect of mediator on protein synthesis in the cytosolic fraction of the cells. Autoradiogram of a 7.5%-acrylamide-0.1% SDS gel analysis of 35 S-methionine labeled cytosolic protein after exposure of the cells of the mediator. 3T3-L1 cells were pulse labeled and the soluble protein was obtained by digitonin as described in Example II. Aliquots (2�105 cpm) of the cytosolic fraction for each time point were applied to the gel and electrophoresed. Lanes 1 and 2, control without exposure to mediator; Lanes 3 and 4, 1 hour exposure to the mediator; Lanes 5 and 6, 3 hours of exposure; Lanes 7 and 8, 6 hours of exposure; Lanes 9 and 10, 20 hours of exposure to conditioned medium from mouse peritoneal exudate cells not exposed to endotoxin; Lanes 11 and 12, exposure of cells to mediator for 20 hours.
FIG. 9 shows the effect of mediator on protein synthesis in the membrane fraction of the cells. Autoradiogram of a 7.5%-acrylamide-0.1% SDS gel analysis of 35 S-methionine labeled membrane protein after exposure of the cells to the mediator. Expreimental design was identical to that described in the legend to FIG. 8. Membrane proteins were obtained by NP-40 extraction as described in Example II. Lanes 1 and 2--control, without exposure to mediator; Lanes 3 and 4, 1 hour of exposure to the mediator; Lanes 5 and 6, 3 hours of exposure; Lanes 7 and 8, 6 hours of exposure; Lanes 9 and 10, 20 hours of exposure of the cells to conditioned medium from mouse peritoneal exudate cells not exposed to endotoxin; Lanes 11 and 12, exposure to mediator for 20 hours.
Friend cells (clone DS-19) were incubated for 96 hours in the absence or in the presence of Me2 SO (1.5 vol %). Conditioned media (80 μl/ml of growth medium) from mouse peritoneal macrophage cultures stimulated or not stimulated with endotoxin (5 μg/ml) were added at the beginning of culture. Cell members were counted with a Cytograf model 6300 and expressed as percent inhibition of the control cells. Cell number in untreated control culture was 3�106 cells/ml. Heme content was determined fluorometrically as described previously (Sassa, S., Granick, S., Chang, C. and Kappas, A. (1975) In Erythropoiesis, ed. by K. Nakao, J. W. Fisher and F. Takaku (University of Tokyo Press, Tokyo) pp. 383-396). Data are the mean of duplicate determinations. The number of trypan blue positive cells assessed by Cytograf counting was 8-10% for all cultures.
______________________________________Cell number   3.0    (� 10-6 /ml)ALA dehydratase         3.00   (nmol PBG/106 cells, h)PBG deaminase 120    (pmol uroporphyrinogen/106                cells, h)Protoporphyrin         0.57   (pmol/106 cells)Heme          520    (pmol/106 cells)______________________________________
Cells were incubated for 96 hours without changing the medium; inducing chemicals and the endotoxin-stimulated macrophage mediator (80 l added/ml of growth medium) time 0. Final concentrations of chemicals were mM for HMBA, 1.3 mM for butyric acid, bmM for hypoxanthine and 0.1 mM for hemin. Assays were performed as described in Example III, infra. Data are the mean of duplicate determinations.
DETAILED DESCRIPTION As disclosed in our above referenced co-pending applications on this subject matter, we have discovered an agent which we identify herein as a mediator substance, that is produced by mammalian cells in response to stimulation by materials we refer to herein as stimulator materials, that characteristically accompany an invasive stimulus, such as bacteria, virus, some tumors, protozoa and other toxins such as endotoxemia. We have observed that the mediator substance causes the metabolism of certain of the cells of the mammal to switch from an anabolic state to a catabolic state. In particular, the mediator substance appears to suppress the activity of anabolic enzymes, such as lipoprotein lipase (LPL), and the other enzymes and inducing agents listed earlier herein. It is theorized that these mediator substance is part of a communications system in mammals, between the immune system and the energy storage tissues of the body. Thus, in response to various invasive stimuli in mammals, such as those listed before, it is theorized that the mediator substance is produced and exert an effect on energy storage tissue such as adipose tissue, muscle, the liver, and the like, of the impending need for energy to combat the invasion. More particularly, the mediator substance may cause these storage tissues to switch from an anabolic to a catabolic state, to facilitate the supply of such energy. If the invasion is of short duration, the mammal can quickly recover and replenish its energy stores; however, if the invasion is of a chronic nature, shock generally manifested by complete energy depletion, cachexia and death, can result.
The invention also relates to methods for detecting the presence of invasive stimuli in mammalian hosts by measuring the presence and activity of the mediator substance. As mentioned earlier, the mediator substance can be used to produce antibodies to themselves in rabbits, goats, sheep, chickens or other mammals, by a variety of known techniques, including the hydridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. The antibody can be isolated by standard techniques and utilized as a test for the presence of the mediator substance in the suspected mammalian hosts.
Further, the antibody or antibodies can be utilized in another species as though they were antigens, to raise further antibodies. Both types of antibodies can be used to determine the presence of mediator substance activity in the mammalian body, particularly in human serum, so as to determine the presence of invasive stimuli such as bacterial, viral, or protozoan infection, or the presence of certain tumors, and to follow the course of the disease. For purposes of the following explanation, the antibody or antibodies to mediator activity, will be referred to as Ab1 the antibody or antibodies raised in another species will be identified as Ab2.
A. Med*+Ab1 =Med*Ab1 B. Med+Ab1 *=MedAb1 *
C. Med+Ab1 +Ab*2 =Med Ab1 Ab2 *
It will be seen from the above, that a characteristic property of Ab2 is that it will react with Ab1. This is because Ab1 raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab2. For example, Ab1 may be raised in rabbits using a mediator as the antigen and Ab2 may be raised in goats using Ab1 as an antigen. Ab2 therefore would be an anti-rabbit antibody raised in goats. For purposes of this description and claims, Ab1 will be referred to as a mediator activity antibody and Ab2 will be referred to as an antibody reactive with a mediator activity antibody or, in the alternative, an "anti-body".
The mediator composition(s) can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotape 14 C, 131 I, 3 H, 125 I and 35 S. The enzyme label can be detected by any of the presently utlized colorimetric spectrophotometric, fluorospectrophotometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galacetosidase, urease, glucose oxidase plus peroxidase, galactose oxidase plus peroxidase and acid phosphatase. U.S. Pat. Nos. 3,654,090; 3,850,752; 4,016,043; are referred to by way of example for their disclosure of alternate labeling material, and materials.
In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of mediator substances in a suspected host. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled mediator or its binding partner, an antibody specific thereto. Another which contain at least Ab1 together with labeled Ab2. Still another will contain at least Ab1 and directions, of course, depending upon the method selected, e.g., "competitive", "sandwich", "DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.
More specifically, a diagnostic test kit for the demonstration of a mammal's reaction to invasive stimuli may be prepared comprising:
(a) a known amount of one mediator substance as described above (or its binding partner) generally bound to a solid phase to form a immunosorbent, or in the alternative, bound to a suitable tag,;
(b) a pharmaceutically acceptable carrier. With the aid of suitable liquids, the antibodies may be used in injection preparations in the form of solutions. These compositions may then be administered to a human in the above manner in shock-reducing amounts to dissipate, if not overcome, the effects of the invasion/shock.
As an adjunct to the development of antibodies and their use in the techniques described above, the present invention extends to methods of treatment of various conditions, such as shock, cachexia etc., that are found to exist as a result of undesirably high mediator substance activity in the mammalian host. In such instance, the method of treatment may include the detection of the presence and activity of the particular mediator substance, and the subsequent administration of the appropriate antibody or antibodies to the host, in amounts effective to neutralize the undesired mediator substance activity.
Conversely, certain adverse conditions in mammals such as obesity, may result from excess anabolic activity. For example, obesity may be caused by undesirably high levels of activity of the anabolic enzymes lipoprotein lipase, acetyl Coenzyme A carboxylase and fatty acid synthetase. The invention accordingly includes a method for treating obesity, comprising administering a mediator substance in an acceptable form, and in an amount effective to assist in restoring proper body weight. Administration of such treatment, however, would be under strict control by a physician, and the amount, manner and frequency of administration of the mediator would be carefully determined and constantly monitored.
In addition to treatment with antibodies raised by a mediator substances, the present invention includes an assay system, for the examination of potential substances, such as drugs, agents, etc. to inhibit the synthesis or activity of a mediator substance. As described earlier, appropriate cell cultures such as the 3T3-L1 and the Friend virus transformed erythroleukemia cells may be initially treated with a particular mediator to inhibit the activity of a particular anabolic actor, after which the potential drug etc. may be added, and the resulting cell culture observed to determine whether changes in the activity of the anabolic actor have taken place. While the foregoing description makes reference to specific cell cultures for the present assay, it is to be understood that the invention is not limited thereto.
TABLE______________________________________               Mediator   MediatorEntity              Production Effect______________________________________Dexamethasone 10-6 M               +          -Aspirin 10-3 M -          -Indomethacin 10-5               -          -Nalaxone 10-5 M               -          -Thyroid Releasing Factor 10-7 M               -          -______________________________________  denotes yes; - denotes no)
The following examples relate to the isolation of the mediator substance, and the observation of its activity, as related to certain anabolic enzymes, etc. A review of the following should lend greater appreciation to the origins and potentials of the present invention. Naturally, however, the specific materials and techniques may vary, as explained earlier, so that the following is presented as illustrative, but not restrictive of the present invention.
EXAMPLE I Isolation of Mediator Activity Compositions A. Mice used in Testing: Male C3H/HeN endotoxin sensitive mice (7-10 wk: 18-25 g) were obtained from Charles River Breeding Laboratory (Wilmington, Mass.). Male C3H/HeJ, endotoxin-resistant mice (7-10 wk: 18-25 g) were obtained from The Jackson Laboratory (Bar Harbor, Maine). Mice were fed ad libitum on Rodent Laboratory Chow (Ralston Purina Co., St. Louis, Mo.) until they were utilized. The chow diet was removed 24 hours prior to each experiment and replaced with a solution of 25% sucrose in water. The animals, once injected, were only allowed access to water. Three to 10 C3H/HeN or C3H/HeJ mice were employed in each experimental group.
The lyphylized filtrate from the ultrafiltration is dissolved in a minimal amount of distilled water, chromatographed on a Sephadex G 50 column (1.6�95 cm), and eluted with PBS (pH 7.4) at a flow rate of 6 ml/hr. Fractions of 3 ml were collected and analyzed for LPL activity. The activity was located in fractions eluting at 170 to 179 ml which corresponds to a molecular weight to about 400 to 1,000 Daltons.
The endotoxin used in the 3T3-L1 cell culture study was obtained as described above. Cell culture media and fetal calf serum were obtained from Gibco Laboratories (Long Island, N.Y.). 3-isobutyl-1-methylxanthine was from Aldrich Chemical (Milwaukee, Wis.), dexamethasone from Sigma Chemical Company (St. Louis, Mo.), and insulin from Eli Lilly Corporation (Arlington Heights, Ill.). Triolein was from Nu Check Prep, Inc. (Elysian, Minn.). Crystalline bovine serum albumin was from Calbiochem-Behring Corporation (LaJolla, Calif.).
I. 3T3-L1 Cell Culture: 3T3-L1 preadipocytes were cultured as previously described [MacKall, et al., J. BIOL. CHEM. 251 at 6462 (1976), and A. K. Student, et al., J. BIOL. CHEM., 255 at 4745-4750 (1980)] in Dulbecoo's modified Eagle's medium (DME medium) containing 10% fetal calf serum. Differentation leading to the adipocyte phenotype was induced by the Student, et al., modification of the method of Rubin, et al., [J. BIOL. CHEM. 253 at 7570-7578 (1978)]. Two days after confluence, the medium was supplemented with 0.5 mM isobutyl-methylxanthine, 1 μM dexamethasone and 10 μg of insulin per ml. Forty-eight hours later, the medium containing isobutyl-methylxanthine, dexamethasone, and insulin was withdrawn and replaced with medium containing insulin at a reduced concentration of 50 ng per ml.
Lipoprotein lipase assays were performed within 30 minutes after the preparation of each sample in duplicate by the method of Nilsson-Ehle and Shotz [J. LIPID. RES. 17 at 536-541 (1976)] with minor modifications. Briefly, 75 μl of enzyme was mixed with 25 μl of substrate containing 22.7 mM[3H]-triolein (1.4 uCi per mole), 2.5 mg per ml of lecithin, 40 mg per ml bovine serum albumin, 33% (V/V) human serum and 33% (V/V) glycerol in 0.27M Tris-HCl, pH 8.1, and incubated at 37� C. for 90 minutes. One milliunit of enzyme activity was defined as the release of one nanomole of fatty acid per minute. The lipase activity in all three compartments was inhibited >90% by addition of 1M NaCl and >80% by omission of serum which is the source of apolipoprotein C-II needed for enzymatic activity.
The medium from the culture of exudate cells not treated with endotoxin had little effect on the lipoprotein lipase activity of 3T3-L1 cells. While the medium from untreated exudate cells elicited some inhibition in the study shown in FIG. 3, Col. B in other similar studies, medium prepared identically had no inhibitory effect. Endotoxin itself also had a negligible inhibitory effect on lipoprotein lipase activity when the amount added was equivalent to that which might remain in the conditioned medium from endotoxin-treated exudate cells; a 19%, 9%, and 0% decrease was observed on medium, heparin-releasable and intracellular compartments, respectively. The decrease was greater (45% in medium, 17% in heparin-releasable, and 11% in the cells) when larger amounts (4.5 times) of endotoxin was employed, as shown in FIG. 3, Column D.
The relationship between the amount of mediator compositions and lipoprotein lipase activity of 3T3-L1 cells was examined by incubating the cells with increasing amounts of the conditioned medium from endotoxin-treated exudate cells for 20 hours at 37� C. Ten μl of conditioned media according to 1.5 ml of culture media was sufficient to cause a substantial decrease in lipoprotein lipase activity, i.e., 57% decrease in the medium, 40% decrease in the heparin-releasable compartment, and 8% decrease in the cells. Enzyme activity was further depressed by increasing the amount of mediator containing medium. When 250 μl were added, a decrease of greater than 95% was observed in all three compartments. The amount of mediator present in conditioned medium varied somewhat from preparation to preparation.
The rate at which lipoprotein lipase activity declines after the addition of the mediators was also investigated. Conditioned medium containing mediators was added at selected intervals, and lipoprotein lipase activity was measured. A reduction of lipase activity was apparent as early as 30 minutes after addition of 3T-3L1 cells. Approximately half of the intracellular enzyme activity was lost after 2.5 hours. After 5 hours of incubation with a mediator, a maximal effect was observed. The amount of enzyme activity in the medium and that on the cell surface were also observed to decrease with a similar time course (data not shown).
The rapid decrease in lipoprotein lipase activity might reflect a competition with insulin since removal of insulin has been shown to lead to a rapid decline in lipoprotein lipase activity in 3T3-L1 cells. However, an attempt was made to reverse the suppressive effect of the mediator by increasing the concentration of insulin in the medium was not successful. For this study, the effect of incubating 3T3-L1 cells with media containing insulin at various concentrations (50 ng/ml to 50 μg/ml) and mediator was assessed for lipoprotein lipase activity. It was found that the inhibitory effect of the mediator on enzyme activity was not changed with increasing insulin concentrations. Even at an insulin concentration 1,000 greater (50 g/ml) than that of standard conditions (50 ng/ml), the inhibition was not reversed.
EXAMPLE II Reasoning that other anabolic activities of the 3T3-L1 cells might be inhibited by the mediator, we studied two key enzymes: (1) acetyl CoA carboxylase; and (2) fatty acid synthetase; for de novo fatty acid biosynthesis. The following example based upon a manuscript in preparation by the inventors herein and coworkers, P. Pekala, M. D. Lane and C. W. Angus, presents evidence that the synthesis of these enzymes are also inhibited by the addition of the macrophage mediator. The results implicate a larger role for the mediator(s) and point to the presence of a communication system between immune cells and energy storage cells of mammals. Presumably, during invasion the immune cells can function as an endocrine system and selectivity mobilize energy supplies to combat the invasion.
A. Materials: Endotoxin (lipopolysaccharide) from E. coli 0127: B8 isolated by the method of Westphal, described supra, was purchased from Difco Laboratories (Detroit, Mich.). Cell culture media and fetal calf serum were obtained from Gibco Laboratories (Grand Island, N.Y.). 3-isobutyl-1-methylxanthine was from Aldrich Chemical (Milwaukee, Wis.); dexamethasone, from Sigma Chemical Company (St. Louis, Miss.); and insulin from Eli Lilly (Indianapolis, Ind.). IgG-SORB was from the Enzyme Center, Inc., (Boston, Mass.). L-[35 S]Methionine (800-1440 Ci/mmol) was from Amersham,. En3 Hance was obtained from NEN, (Boston, Mass.). Antiserum to fatty acid synthetase was kindly provided by Dr. Fasal Ashmad of the Papanicolau Cancer Research Institute, Miami, Fla.
The cells (4�105 cells per cm2d) were incubated in serum-free RPMI-1640 medium for 3 hours after which nonadherent cells were removed by washing 3 times with medium. Cells adhering to the dish were primarily macrophages. These cells were further incubated in serum-free RPMI-1640 medium in the presence or absence of 10 g per ml of endotoxin. After 24 hours, the culture medium was removed and centrifuged at 1,000�g for 5 minutes at 4� C. The supernatant of conditioned medium obtained from cells exposed to endotoxin was assayed and found to contain the mediator substance that lowers LPL in 3T3-L1 cells.
E. Labeling of Cellular Proteins: A 6-cm plate containing induced 3T3-L1 cells was washed twice with 5 ml of methionine-free medium and incubated for 1 hour with 2 ml of the same medium containing 0.5 mCi of L-[35 S]-methionine during which period the rate of [35 S]-methionine incorporation into cellular protein was linear. The medium was removed, the cell monolayer washed twice with phosphate-buffered saline, ph 7.4, and the soluble cytosolic proteins released by the digitonin method of Mackall, et al., supra. The remainder of the cell monolayer containing the membranous fraction was then scraped into 2.0 ml of 100 mM HEPES buffer, pH 7.5, containing 0.5% of the nonionic detergent NP-40 and 1 mM phenylmethylsulfonylfluoride. After trituration in a Pasteur pipet, the suspension was centrifuged at 10,000�g for 10 minutes at 4� C., and the supernatant saved.
[35 S]-methionine incorporation into acid insoluble material was determined by adding 20 μl of digitonin or NP-40 released material to 0.5 ml of ice cold 20% TCA with 25 μl of 0.5% bovine serum albumin added as carrier. After sitting at 4� C. for 1 hour, the mixture was centrifuged at 2,000�g for 5 minutes. The pellet was incubated in 0.5 ml of 1M NH4 OH at 37� for 30 minutes. The protein was reprecipitated on addition of 5.0 ml of ice cold 10% TCA and filtered on Whatman GF/C filters. The filters were extracted with diethyl ether and the amount of radiolabel determined.
F. Immunoadsorption Electrophoroesis: Aliquots of the soluble [35 S]-methionine-labeled proteins from the soluble (digitonin released) fraction of the cell monolayer were made 1 mM in PMSF and 0.5% in NP-40 detergent and then added to 5 l of either antisera specific for acetyl CoA carboxylase, or fatty acid synthetase, After 2 hours at 25� C., 100 μl of 10% IgG-SORB were added and the labeled enzymes isolated from the mixture by the method of Student, et al., supra. Polyacrylamide-SDS gels were run according to the method of Laemmli, and prepared for fluorography by use of En3 Hance according to the manufacturer's instructions.
G. Results--Effect of Mediator on Acetyl CoA Carboxylase and Fatty Acid Synthetase: To examine the effect of the mediator substance on the activities of acetyl CoA carboxylase and fatty acid synthetase enzymes, 3T3-L1 cells were exposed to conditioned medium from mouse peritoneal exudate cells cultured in the presence of endotoxin. After incubation of the 3T3-L1 cells with the mediator for 3, 6 and 20 hours, acetyl CoA carboxylase and fatty acid synthetase activities were determined on a digitonin released cytosolic fraction of the cells (FIG. No. 4). The activity of both enzymes decreased over the 20-hour period to approximately 25% of the initial values.
To determine if the loss in activity of the two enzymes was a result of a direct effect on protein synthesis, 3T3-L1 cells were incubated with conditioned medium from cultures of endotoxin-treated exudate cells for 3, 6, and 20 hours. During the final hour of incubation, the cells were exposed to a pulse of 35 S-methionine. Following the pulse, 35 S-methionine labelled acetyl CoA carboxylase and fatty acid synthetase were isolated from the digitonin releasable cytosolic fractions by immunoadsorption. Identification was accomplished by SDS-polyacrylamide gel electrophoresis and fluorography (FIGS. No. 5A and 6A). The decreased incorporation of 35 S-methionine into immunoadsorbable acetyl CoA carboxylase and fatty acid synthetase with respect to time following exposure to the mediator is readily observed. Densitometric scanning of the autoradiograms (FIGS. No. 5B and 6B) indicated that after 20 hours of exposure to the mediator, the amount of 35 S-methionine incorporated into fatty acid synthetase and acetyl CoA carboxylase were decreased by 80% and 95% respectively. These results are consistent with the concept that the mediator depresses the activity of acetyl CoA carboxylase and fatty acid synthetase by interfering with the synthesis of the enzyme.
H. Effect of Mediator on Protein Synthesis in General: The observed effect on acetyl CoA carboxylase and fatty acid synthetase could be explained by a general inhibition of protein synthesis by the mediator. To examine this possibility, the effect of mediator on amino acid incorporation into protein was investigated. 3T3-L1 cells were incubated for various periods of time with conditioned medium obtained from mouse peritoneal exudate cells cultured in the presence of endotoxin. 35 S-methionine incorporation into soluble and membrane associated protein was determined after 1, 3, and 6 hours of exposure of the cells to the added factor. When 3T3-L1 cells were exposed to conditioned medium from mouse peritoneal exudate cells that were cultured in the absence of endotoxin, no effect on 35 S-methionine in incorporation into acid insoluble protein was observed. However, as seen in FIG. No. 7, 35S-methionine incorporation into TCA precipitable material in the soluble fraction (Digitonin releasable protein) increased approximately 10% in the first 3 hours with no further change observed, while a 50% decrease was observed for label incorporation into acid insoluble material in the membrane fraction (NP-40 solubilized protein). Analysis of 35 S-methionine labeled proteins following exposure to the mediator was accomplished utilizing SDS-gel electrophoresis. The pattern of the autoradiogram of the soluble proteins obtained on digitonin treatment and those solublized by NP-40 of the 3T3-L1 cells are shown in FIGS. No. 8 and 9. Closer inspection of FIG. No. 8 reveals the gradual disappearance with time following the addition of the mediator of a protein band with a molecular weight of 220,000 Daltons, while another band appears at approximately 18,000. In addition to these major changes, another new protein appears at approximately 80,000 while a second protein of 50,000 disappears.
I. Analysis. The mediator appears to decrease enzymatic activity by suppressing the synthesis of the enzymes. The effect on protein synthesis appears to be quite specific as there are no gross perturbations of the protein patterns observed on the autoradiograms (FIGS. No. 8 and 9). In response to the mediator, the synthesis of several proteins is inhibited or induced. It was possible by immunoprecipitation to identify fatty acid synthetase and acetyl CoA carboxylase (M.W. 220,000) as two proteins whose synthesis is inhibited by the mediator. The identification of the other proteins that are modulated by the mediator is not possible at present, although lipoprotein lipase is a potential candidate for the 50,000-Dalton protein that appears. The nature of proteins that are induced in response to the mediator and the mechanism for the modulation of specific protein synthesis are deserving of further improvement investigations.
EXAMPLE III In this series of investigations, also embodied in an unpublished manuscript in preparation by the inventors herein, and co-worker Shigeru Sassa, we sought to determine whether the macrophage mediator(s) observed in Examples I and II exerted any effect upon red blood cell synthesis. We reasoned that, as anemia is commonly observed in mammals afflicted with chronic infections, and that as regeneration of the red cell mass constitutes a potential drain on energy and amino acids, the body in response to acute invasion may interrupt erythroid development in similar fashion and perhaps by the same mechanism observed with respect to the anabolic enzymes lipoprotein lipase, acetyl Coenzyme A carboxylase and fatty acid synthetase, that effect adipocytes.
To evaluate this hypothesis, we examined the effects of endotoxin-induced factor(s) from mouse macrophages on the cellular proliferation and differentiation of a model erythroid progenative cell--the Friend virus-transformed erythroleukemia cells (See Friend, C. et al and Marks, P. A. et al., supra.). In this model system, cells can be induced to differentiate and form hemoglobin in response to a number of inducers, such as dimethylsulfoxide, (Friend, C., et al supra.), hexamethylenebisacetamide (Reuben, R. C. et al, PROC. NATL. ACAD. SCI., U.S.A., 73: 862-866), butyric acid, (Leder, A. et al (1975) Cell 5: 319-322), and hypoxanthine (Gusella, J. F. (1976) Cell 8: 263-269). This example presents evidence that a macrophage mediator(s) can inhibit the growth and differentiation of erythroid committed cells, but has less effect on uncomitted stem cells and practically no effect on fully differentiated erythroid cells.
A. Materials: Endotoxin (lipopolysaccharide) from E. coli 0127: B8 isolated by the method of Westpal (described supra.), was purchased from Difco (Detroit, Mich.). A modified F12 medium was prepared in our laboratory (Sassa, S. et al, J. BIOL. CHEM. 252: 2428-2436 (1977)). Fetal bovine serum was purchased from GIBCO (Grand Island, N.Y.). Dimethylsulfoxide (Me2 SO) was a product of Eastman Organic Chemicals (Rochester, N.Y.). Butyric acid and hypoxanthine were obtained from Sigma Chemical Company (St. Louis, Mo.). Hexamethylenebisacetamide (HMBA) was kindly provided by Dr. R. C. Reuben, Merck Sharp & Dohme Research Laboratories (Rahway, N.J.).
B. Cell Culture: Murine Friend-virus transformed erythroleukemia cells (clone DS-19) were cultivated in modified F12 medium supplemented with 10% heat inactivated fetal bovine serum as described previously (Sassa, S., Granick, J. L., Eisen, H. and Ostertag, W. (1978) In In vitro Aspects of Erythropoiesis, ed. by Murphy, M. J. Jr. (Springer-Verlag, N.Y.) pp. 268-270).
C. Preparation of the Endotoxin-Stimulated Conditioned Medium From the Culture of Mouse Exudative Cells: Isolation of peritoneal exudate cells from NCS mice (25-33 g from the Rockefeller University Breeding Colony) and preparation in vitro of an endotoxin-stimulated conditioned medium were carried out as described (in Example I, above). Briefly, peritoneal exudate cells were isolated from mice treated with sterile Brewer's thioglycollate medium obtained from Difco Laboratories (Detroit, Mich.), in an amount of 3 ml per mouse, 6 days prior to harvest. The cells were incubated in serum-free RPM1-1640 medium for 3 hours, after which non-adherent cells were rinsed off by washing three times with medium. Cells adhering to the dish were primarily macrophages (Kawakami et al., PROC. NATL. ACAD. SCI., USA 79:912-916; Edelson, P. S. et al., J. EXP. MED., 142: 1150-1164 (1975)).
D. Induction of Erythroid Differentiation: Two types of incubation protocols were used to assess erythroid differentiation of Friend cells. In certain experiments illustrated in FIGS. 10-13, the cells (5�104 cells/ml) were incubated at 37� C., in 5% CO2 in humidified air for 18 hours. The inducing chemicals, e.g. Me2 SO, HMBA, butyric acid, hypoxanthine or hemin were added with or without macrophage mediator(s) and cultures were incubated for 96 hours without changing the growth medium. In other experiments such as those with results illustrated in FIG. 14, the cells (105 cells/ml) were incubated for 18 hours, then Me2 SO and the macrophage mediator were added as above. The cultures were maintained at 2�105 cells/ml by diluting the cell suspension daily with fresh medium containing the chemical inducer with or without the macrophage mediator. This procedure required more macrophage mediator than the first experimental procedure, but made it possible to examine the effect of mediator on rate of cell growth while cells were growing logarithmically at a constant rate (Chang, C. S. et al; J. BIOL. CHEM. 257: 3650-3654 (1982)).
E. Determination of Heme Content and Assays on the Activities of Enzymes In the Heme Biosynthetic Pathway: The concentration of heme in cells was determined by a fluorometric assay of prophyrin derivatives after the removal of iron (Sassa, S., Granick, S., Chang, C. and Kappas, A., In Erythropoisis, ed. by K. Nakao, J. W. Fisher and F. Takaku (University of Tokyo Press, Tokyo, Japan (1975) pp. 383-396). Cells containing hemoglobin were stained with benzidine and counted using a Cytograf model 6300A (Sassa, S., Granick, J. L., Eisen, H., and Ostertag, W., Supra.). Assays of aminolevulinic acid (ALA) dehydratase and porphobilinogen (PBG) deaminase were carried out by methods described previously (Sassa, S., Granick, J. H., Eisen, H., and Ostertag, W., Supra.).
F. Effects of the Macrophage Mediator on the Growth and Differentiation of Uninduced Friend Cells: Conditioned media from macrophage cultures incubated with or without endotoxin inhibited the growth of untreated Friends cells by approximately 35% (FIG. 10, Part A.). When these cells were incubated simultaneously with 1.5% Me2 SO, control conditioned medium which had not been exposed to endotoxin inhibited the cell growth by �42% while endotoxin-stimulated conditioned medium inhibited the growth of �60% (FIG. 10, Part B).
Heme content in these cells treated with endotoxin-stimulated or non-stimulated conditioned media was not appreciably different from that found in untreated cells, indicating that the conditioned medium by itself does not affect the erythroid differentiation of Friend cells (FIG. 10, Part B). In contrast, incubation of cells with Me2 SO and endotoxin-stimulated conditioned medium led to a significant decrease (�40%) in the heme content in the cell (FIG. 10, Part B).
G. Dose Dependent Inhibition of Cell Growth and Differentiation By the Macrophage Mediator: When Friend cells were incubated simultaneously with 1.5% Me2 SO and the endotoxin-stimulated macrophage mediator, the rate of cell growth was progressively inhibited when increasing amounts of the mediator were added to the culture (FIG. 11, Part A). An inhibitory effect of the mediator on cell growth could be detected at the lowest concentration examined (1.12 vol. % added to growth medium). At the highest concentration (8 vol. %), the mediator inhibited cell growth by �60% compared with that of the control Me2 SO-treated culture (FIG. 11, Part A). The decrease in cell number was not due to cell death since the number of dead cells as accessed by the Trypan Blue exclusion test (Paul J. In Cell Culture) was similar (�8%) for untreated controls and cultures treated with the stimulated conditioned medium. Endotoxin itself (up to 15 μg/ml) exhibited no inhibitory effect on the growth of Friend cells either in the presence or in the absence of Me2 SO (data not shown). These findings indicate that the endotoxin-stimulated macrophage mediator interferes with the growth of Me2 SO-treated cells more than that of untreated cells and suggest that erythroid committed cells may be more sensitive than uncommitted stem cells to the action of the stimulated macrophage mediator.
H. Delayed Addition of the Endotoxin-Stimulated Macrophage Mediator on Erythroid Differentiator: When the endotoxin-stimulated conditioned medium was added to Me2 SO-treated cultures at various times, it was found that the effect of the macrophage mediator on cell growth was gradually lost (FIG. 12).
I. Effects of The Endotoxin-Stimulated Macrophage Mediator On Erythroid Differentiation of Friend Cells Induced by HMBA, Butyric Acid, Hypoxanthine or Hemin: In order to examine whether or not the inhibitory effect of the endotoxin-stimulated macrophage mediator on erythroid committed cells is specific for Me2 SO-induced differentiation, we examined the effect of the macrophage mediator on cells which were incubated with either HMBA, butyric acid, hypoxanthine, or hemin. We found that the endotoxin-stimulated macrophage mediator markedly inhibited the growth of cells incubated with HMBA, butyric acid or hypoxanthine, but not the growth of hemin-treated cells (FIG. 13, Part A). Similarly, the endotoxin-stimulated mediator inhibited the erythroid differentiation induced by HMBA, butyric acid or hypoxanthine, but not that induced by hemin treatment (FIG. 13, Part B).
Under these conditions of culture, the cells maintain a continuous logarithmic growth at a constant rate (Chang,). C. S. et al supra.) The total number of cells that would have formed from the original untreated control culture was 82�106 cells/ml after 96 hours of incubation (FIG. 14). The addition of the macrophage mediator significantly inhibited (�70%) cell growth. The addition of Me2 SO to the cultures yielded 42�106 cells/ml. This decrease probably reflects the growth cessation which is associated with terminal erythroid differentiation of these cells. (Chang, C. S. supra.; Lo, S.C., Aft., R. and Mueller, G. C., Cancer Res. 41: 864-870 (1981)). Combined addition of Me2 SO and the macrophage mediator produced the most profound growth inhibition (�90%) of these cells. Heme content in cells treated with the mediator alone was not appreciably affected while the combined treatment with the mediator and Me2 SO brought about �40% inhibition of heme formation.
K. Analysis: The mediator substance under study appears to potently inhibit the growth and erythroid differentiation of mouse Friend-virus transformed cells. Conditioned medium from cultures not exposed to endotoxin had some inhibitory effects, but the effect of the endotoxin-stimulated conditioned medium is significantly greater in inhibiting the growth and differentiation of Friend cells. Endotoxin itself had no effect on either cell growth or differentiation.
Further, the effect of the mediator appears to be specific to certain stages of erythroid progenitor cells, in that the macrophage mediator inhibited the growth and erythroid differentiation of uncommitted stem cells more than that of erythroid committed cells which were induced by treatment with Me2 SO, HMBA, butyric acid or hypoxanthine. The inhibitory effect of the macrophage mediator on cell growth was more pronounced in cells growing logarithmically at a constant rate. Hemin treatment of Friend cells is known to cause erythroid cells maturation leading to the appearance of hemoglobinized cells but without accompanying the commitment of undifferentiated stem cells to the erythroid precursor cells (Gusella, J. F., Weil, S. C., Tsiftsoglon, A. S., Volloch, V., Neuman, J. R. and Housman, D. (1976) Blood 56: 481-487). Interestingly, the endotoxin-stimulated macrophage mediator also had very little effect on the growth and differentiation of Friend cells in the presence of hemin.
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