Leukocytic Endogenous Mediator Research Articles

From the Obscure and Mysterious Acute Phase Response to Toll-Like Receptors and the Cytokine Network

The discovery of C-reactive protein (CRP) almost 80 years ago in the blood of patients with various infectious and inflammatory diseases initiated studies on the mysterious biological phenomenon called “the acute phase response” (APR). The complex metabolic alterations accompanying APR include changes in the plasma concentration of the majority of liver-produced proteins – acute phase proteins (APPs). The search for mediators released from the site of injury and able to stimulate hepatocytes led to the discovery of “Leukocytic Endogenous Mediator” (or ”Endogenous Pyrogen”), initially identified with interleukin-1. Only in 1987 was the main factor able to induce the majority of symptoms of APR discovered and named interleukin-6 (IL-6). The current paradigm assumes that APR is elicited by numerous proinflammatory cytokines, the principal role being played by the IL-6-family. The last decade has brought about significant progress in understanding the initiation of the acute phase response (recognition of pathogens by Toll-like receptors), interaction of cytokines and their receptors (origin and importance of soluble cytokine receptors, construction of hypercytokines by protein fusion), the mechanism of multi-step signal transduction from the plasma membrane to nuclear transcription factors (including the role of MAP kinases), as well as elucidation of the role of receptor cross-talk in cytokine networks in health and disease. Genomic techniques indicate that hundreds of genes participate in the development of APR. The data presented here emphasize the growing importance of APR for clinical medicine and confirm the close relationship between acute phase response and innate immunity. Keywords: Acute phase proteins, pro-inflammatory cytokines, cytokine pleiotropy and redundancy, soluble receptors and engineered hypercytokines, Toll-like receptors and pathogen recognition, cytokine networks and receptor cross-talk

Kinetics and mechanisms of release of serum proteins of intoxicated rat livers implanted into guinea pigs

Abstract We examined the organic chemistry, kinetics, and mechanisms of release of rat serum proteins by aseptic rat donor livers (DL) pathologically intoxicated by CCl4, alloxan, or cholera toxin implanted into the circulatory systems in parallel with the host livers (HL) of guinea pigs (GP). This implant model was biologically functional as hepatocytes of the DL released rat plasma proteins into the host's extracellular compartment (ECC) as monitored in GP serum samples collected during 4 h of the experiment. Furthermore, rat DL hepatocytes retained an in vivo preimplantation pattern of protein biosynthesis acquired in response to a regulatory mechanism most likely involving leukocytic endogenous mediator (LEM), kinins, or platelet activating factor (PAF), and consequently secreted the entire spectrum of acute-phase reaction (APR) serum proteins into the ECC of the host GP. Therefore, the impetus to synthesize and release APR proteins into GP circulation by intoxicated rat liver originated within and was retained by injured rat hepatocytes.

Interleukin 1 and interferon-gamma: cytokines that provide reciprocal regulation of macrophage and T cell function.

Interactions between macrophages and T cells are symbiotic, since optimal functions of both cell types require interchange of soluble mediators. Upon activation, macrophages release interleukin-1 (alias lymphocyte activating factor, leukocytic endogenous mediator, and endogenous pyrogen), a family of molecules with multivarious biological effects, ranging from induction of fever and the acute phase response to lymphocyte activation and concomitant release of interleukin-2. Interleukin-2 induces activation and replication of several subsets of precursor lymphocytes, including cytotoxic T cells, lymphokine-activated killer (LAK) cells, and natural killer (NK) cells, and enhances their cytotoxic activity for tumor cells. Both interleukin-2 and leukotrienes enhance production of immune interferon (interferon-gamma) by activated T cells. Aside from antiviral activity, interferon-gamma produces a number of immunomodulatory effects, including macrophage activation (Ia induction, antimicrobial effector function, and activation of oxidative metabolism) and augmentation of NK function. Expression of Ia on accessory cell membranes is required for the initiation of many antigen-specific, T-dependent immune responses. Interferon-gamma also synergizes with a variety of microbial agents to augment macrophage tumoricidal function and enhance interleukin-1 secretion. The production of interferon-gamma appears to have a critical role in feeding back the cascade of interleukins in a loop of amplification. Both interleukin-1 and interferon-gamma modulate release of arachidonate metabolites in various cells. This cascade of cytokines, in collaboration with arachidonate oxygenation products, regulates immunity and sets the stage for many of the events underlying inflammation. Various anti-inflammatory drugs and immunopotentiators appear to act by modulating cytokine pathways.

Fever and changes in plasma zinc and iron concentrations in the goat: The role of leukocytic pyrogen

In goats with trypanosomiasis ( T. vivax or T. congolense) no marked fall in plasma zinc concentration was seen despite high temperature peaks, whereas plasma concentrations of iron tended to undergo some decline. In goats infected with Ehrlichia phagocytophila, there was a marked decline in plasma zinc and iron to low values on the 3rd and 4th day, respectively. Circulating endogenous pyrogen (EP) or leukocytic endogenous mediator (LEM) could not be detected in plasma from febrile goats with tick-borne fever. The intravenous injection of leukocytic pyrogen (LP) in kids caused characteristic monophasic febrile reactions, whereas no significant changes in plasma trace metals were found. So, previous evidence purporting to show that LP is similar to or may be identical with LEM is demonstrably inconclusive. Intravenous injection of E. coli lipopolysaccharide (LPS) or staphylococcal enterotoxin B (SEB) induced fever and lowering of plasma zinc and iron concentrations. The decrease in those trace metal values was more persistent in goats given SEB than in those given E. coli LPS. After intramammary infusion of SEB or E. coli LPS, fever and significant decreases in plasma zinc and iron concentrations were observed but no clear relationship was found between the temperature responses and the alterations in plasma trace metal concentrations. Furthermore, the decrease in plasma iron concentration developed more rapidly in goats given SEB than in those given E. coli LPS, whereas the decrease in plasma zinc concentrations in the former was more delayed. These data support the theory that the concentrations of zinc and iron in plasma are regulated by different mechanisms, whereas febrile reactions are mediated by another type of endogenous protein.

The Numerous Postulated Biological Manifestations of Interleukin-1

Abstract Over the past 25 years there has been an ever-expanding interest in the mediators that control the immune response [98,133], One of these is produced by macrophages and was first called “lymphocyte activating factor” [49–51] but is now designated “interleukin-1” (IL-1) [118]. As the physical characteristics of IL-1 began to be described, it was noted that they were similar to those of endogenous pyrogen (EP) [128] [151] and leukocytic endogenous mediator (LEM) [85]. Since then, many of the biochemical changes that occur in the host during infection, inflammation, or neoplasia have been attributed to this unique family of monokines. In this review, the family of monokines that has similar physical and biological properties will be referred to as EP/LEM/IL-1. When only one biological activity was measured, the designation will be for that activity. It will be the purpose of this review to examine the evidence that suggests that EP/ LEM/IL-1 has numerous biological activites and to describe its chemical and biological properties, species specificity, and possible sites of action. Finally, I will attempt to address the issue of why there might be a closely related group of mediators with such a broad range of biological activities.

Interleukin-1.

For the past 30 years, considerable experimentation on the mechanisms of host responses to infection has centered on soluble products derived from phagocytic cells. The biologic activities of some of these products include fever mediated by endogenous pyrogen (EP) and induction of acute-phase responses by leukocytic endogenous mediator (LEM), EP and LEM have been characterized and purified and appear to be closely related, if not identical, molecules. Lymphocyte-activating factor (LAF), a recently described polypeptide that acts on lymphocytes, shares many of the physical properties of EP and LEM; when incubated with lymphocytes, purified EP/LEM is indistinguishable from LAF. The term interleukin-1 (IL-1) is now used to describe LAF, EP, and LEM as a single molecule or as a family of closely related molecules, although at present there is no known sequence analysis of EP, LEM, or LAF. In this review, experimental and clinical data are presented that link mediation of host responses to infection and inflammation to the production and activity of IL-1. Cell sources and inducers of IL-1 are discussed, as are its chemical nature and mechanisms of action. In addition, the importance of IL-1 and its effects on host defense mechanisms are presented. For example, how IL-1-mediated responses, such as elevated temperature, lymphocyte activation, and systemic metabolic changes, alter the host as well as the invading microbe are considered. The conclusions of this review are (1) that IL-1 is a key mediator of host responses to microbial invasion, (2) that IL-1 represents a true hormone produced during infection and inflammation, and (3) that its biologic activities account for several aspects of the acute-phase reaction.

Depressed response of plasma iron and zinc to endotoxin and LEM in STZ-diabetic rat.

Laboratory and epidemiological evidence indicate that the enhanced flux of iron and zinc from the plasma to the storage compartments, such as liver, serves as a protective host response to combat infection. Studies were performed to determine the status of this nonspecific immune response in the diabetic animal, since it is commonly held that the diabetic has an increased incidence and susceptibility to infection. Normal rats and rats previously rendered diabetic by streptozotocin (STZ) were injected with either saline or Escherichia coli endotoxin, and plasma levels of zinc, iron, and copper were monitored 8 hr thereafter. Diabetic rats reduced their plasma zinc and iron levels by 35 and 25%, respectively, in response to endotoxin injection whereas control rats had a 70% decrease in zinc and a 46% depression in iron. Insulin administration to the diabetic rats restored the ability to decrease plasma zinc and iron to the same degree as control rats. Plasma copper did not change in any group. Further investigation suggested that the defect in trace metal response occurred after the secretion of leukocytic endogenous mediator (LEM) in the inflammatory response pathway. It is concluded that STZ-diabetic rats have a diminished ability to decrease plasma zinc and iron in response to endotoxin, and that this defect is due to an ineffective response of target tissues to the effects of leukocytic endogenous mediator. Furthermore, it is postulated that the hyperinsulinemia associated with the stress of infection functions to lower plasma zinc and, possibly, iron, thereby allowing the host to better combat infection.

Turnover in the transferrin iron pool during the hypoferremic phase of experimental Neisseria meningitidis infection in mice.

Mouse transferrin was used to specifically label the plasma transferrin iron pool for studies of iron kinetics in normal mice and infected mice during the hypoferremic phase of experimental meningococcal infection. The plasma transferrin iron pool of normal mice was found to be very dynamic, with a half-life of iron in the pool of 0.7 h. Iron left the plasma pool, entered the bone marrow, and was released into the blood in erythrocytes. Iron from the transferrin pool also entered the liver and spleen and was presumably in the reticuloendothelial system components of these organs. Most of the iron that had been supplied as transferrin iron was found in erythrocytes by 48 h after injection. Studies with mice infected with Neisseria meningitidis strain M1011 revealed similar kinetics for transferrin iron. There was no redistribution of iron within the various iron pools as a result of infection. Iron turnover in the plasma transferrin pool during the hypoferremic phase was similar to control rates, and iron leaving the pool entered its normal erythroid compartments. The lack of accelerated turnover of plasma iron and the finding that plasma iron was not rerouted to storage compartments during the hypoferremic phase provided good evidence that lactoferrin and leukocytic endogenous mediator were not directly involved in redirecting transferrin iron. Our evidence has implicated an impaired return of reticuloendothelial system-processed iron to the transferrin pool during the hypoferremic response. This appears to be a logical point in the erythroid iron cycle for host-mediated iron sequestration, as the reticuloendothelial system is involved in iron storage and may regulate iron levels in the plasma transferrin pool under normal conditions.

Characterization of a leukocyte-derived endogenous mediator responsible for increased plasma fibrinogen.

Fibrinogen has been the plasma protein most frequently studied after tissue injury. This report presents evidence that leukocytic endogenous mediator (LEM) from macrophages promotes fibrinogen synthesis. LEM has a molecular weight of 13,000-16,000, an isoelectric point (pI) at pH 7.3, is heat labile, and is inactivated by trypsin or sulfhydryl reactive agents. LEM not only promotes increased synthesis of acute phase proteins, but also causes increased neutrophilia and alterations in metal metabolism. There is considerable evidence that LEM may be the same protein as endogenous pyrogen and Interleukin 1 (IL-1). There was no increase in plasma fibrinogen when endotoxin was injected in C3H/HeJ mice; however, this strain of mice responded the same as normal mice to injections of LEM. This provides further evidence that LEM is the endogenous mediator for acute phase protein synthesis during tissue injury. The half-life of LEM is still circulation following its iv injection into rats was less than 10 minutes. There is still considerable doubt about the mechanism LEM uses in promoting increased hepatocyte synthesis of fibrinogen. Some evidence indicates a direct action of LEM upon the hepatocyte, whereas other data suggest an indirect role through other mediators or the central nervous system. In addition to LEM with pI of 7.3, there are proteins with a pI near 5 that will increase plasma fibrinogen. These proteins also have a molecular weight between 13,000 to 16,000 but do not have essential sulfhydryl groups. These proteins also have pyrogenic and IL-1 activities. LEM shows a limited amount of species specificity. For example, the pI 7 LEM prepared from human monocytes or rabbit peritoneal leukocytes will increase plasma fibrinogen in rats, mice, and rabbits; but the pI 5 LEM from rabbits is inactive in rats.

Hepatic metallothionein induction in inflammation.

Numerous chemically distinct phlogistic substances have been shown to induce hepatic metallothionein-Zn (MT) accumulation when administered to rats. These findings suggest that induction of this cysteine-rich metalloprotein occurs through the action of some common mediator(s). Possible mediators include substances such as leukocytic endogenous mediator (LEM) and/or hormones known to influence hepatic protein synthesis. Studies were performed to examine further the mechanism(s) and potential mediators involved in endotoxin-induced MT accumulation. Additionally, the studies were performed to determine the possible involvement of genetic factors, which reportedly influence LEM production, in the induced MT response. Endotoxin (ET) was administered ip to rats and to EP-resistant, C3H/HeJ, and susceptible, C3Heb/FeJ, stains of mice. ET induced hypozincemia, hyperglucagonemia, and increased MT concentrations in rats. ET induced hypozincemia and MT accumulation to the same extent in both strains of mice. The induction of tolerance in rats to Zn depressing activity of ET also prevented hyperglucagonemia and additional accumulation of MT. Results suggest that glucagon, but not LEM, may be a common mediator in MT response during inflammatory stress.

Stereotypic responses to infection and inflammation: probable activation of phagocytic cells.

There is a sequence of Stereotypie metabolic changes in rats subjected to inflammation or infection. In each stress, rats respond with increases in body temperature and plasma insulin and with decreases in plasma zinc, ketone bodies, and free fatty acids. The data suggest that infection or inflammation causes an activation of phagocytic cells and also that leukocytic endogenous mediator, when injected into rats, causes some of the same alterations. Rats doubly vagotomized respond to infection in the same fashion as intact rats.

An in vitro bioassay for leukocytic endogenous mediator(s) using cultured rat hepatocytes.

Primary cultures of adult rat hepatocytes were used to assay for the presence of leukocytic mediator(s) (LEM), a neutrophil derived protein(s) capable of stimulating the synthesis of acute-phase plasma proteins when injected into rats. In the presence of physiological concentrations of dexamethasone (40 mM), the hepatocytes secreted a variety of plasma proteins as demonstrated by crossed immunoelectrophoresis. The addition of LEM to hepatocytes increased the secretion of several acute-phase related plasma proteins, including fibrinogen and hepatoglobin, and decreased albumin secretion. These results mimic the acute-phase response observed in the intact animal. Fibrinogen secretion was used as a quantitative marker for determining LEM activity. The rate of fibrinogen secretion depended upon both the concentration of dexamethasone and LEM present during a given 24-h assay period. One unit of LEM activity is defined as that concentration of LEM capable of producing a 50% maximal stimulation of fibrinogen secretion.

Rate of clearance of circulating leukocytic endogenous mediator in the rat.

AbstractThe rate of clearance of leukocytic endogenous mediator from the circulation of the rat was determined by measuring the rate of disappearance of activity from the plasma. Leukocytic endogenous mediator was injected iv into groups of rats and the plasma was periodically sampled for the activities that would lower plasma iron, increase plasma fibrinogen, or release bone marrow neutrophils. These activities were assayed by injecting the plasma into groups of normal rats. The t1/2 's for the clearance of leukocytic endogenous mediator ranged between 6 and 10 min.

Leukocytic endogenous mediator in Crohn's disease.

Reduced concentrations of plasma zinc associated with elevated levels of serum leukocytic endogenous mediator activity were found in 17 patients hospitalized with acute exacerbation of Crohn's disease. Neither the decrease in plasma zinc nor the increase in leukocytic endogenous activator activity in nine outpatients with quiescent disease was significant. Leukocytic endogenous mediator activity may be increased in inflammatory conditions as well as in microbial infections. Although zinc deficiency may be common in patients with Crohn's disease, exclusive reliance on circulating zinc levels to assess zinc nutriture in active Crohn's disease may be misleading.

Effect of human monocyte pyrogen on plasma iron, plasma zinc, and blood neutrophils in rabbits and rats.

SummaryStimulated human monocytes produce two physically distinct pyrogens. Both of these partially purified pyrogens will lower plasma iron and zinc and increase blood neutrophils when injected into rats or rabbits. When equivalent pyrogenic doses were injected, the two pyrogens gave quantitatively similar leukocytic endogenous mediator activities (i.e., granulocytosis and lowering of plasma iron and zinc). These results, therefore, provide evidence that leukocytic endogenous mediator can be produced by monocytes as well as by granulocytes.

Effects of leukocytic endogenous mediator on hemopexin, transferr, and liver catalase.

SummaryElevations in the plasma levels of hemopexin and fibrinogen were found following injection of leukocytic endogenous mediator (LEM) into rats. Liver catalase and plasma transferrin were decreased following injection of LEM. Greater amounts of LEM were required to depress liver catalase and plasma transferrin than were required to elevate hemopexin or fibrinogen. Multiple injections of LEM at 8-hr intervals were found to be more effective for depression of liver catalase and plasma transferrin than a single large dose of LEM.

Changes in body balances of nitrogen and other key nutrients: description and underlying mechanisms

This paper represents an attempt to explain the apparent differences in host metabolism between prolonged fasting or starvation and the anorectic state which accompanies severe infection. The differences are discussed in terms of a purposeful redistribution of amino acids from peripheral to visceral tissues for various aspects of host defense, in particular for the synthesis of acute phase globulins. Phagocytosis is the proposed trigger for this redistribution of amino acids and increased acute phase globulin synthesis, as well as for an array of physiological alterations which include, but are not necessarily limited to, fever, decreased plasma zinc and iron, increased plasma copper (ceruloplasmin), glucagon, and insulin. A concomitant (and perhaps consequence) of phagocytosis is the release of factors such as pyrogen and leukocytic endogenous mediator. These mediators are capable of initiating a wide range of metabolic alterations associated with infection. The panoply of metabolic alterations which occur after phagocytosis and before the development of immunity is presented as a necessary part of an integrated host response to inflammatory stress. The redistribution of trace metals and the increased acute phase globulin synthesis appear to provide both positive and negative feedback: 1) by delimiting tissue damage occasioned by the infection or by phagocytes in response to infection; 2) through increase of phagocytic capacity, in part by promoting granulopoiesis; and 3) by aiding in the inactivation of the microorganism and in the development of immunity. Consonant with the suggested import of the amino acid redistribution and increased acute phase globulin synthesis is the fact that, contrary to what is the case in the fasting state, plasma insulin is increased and ketosis is inhibited in severely septic animals and patients; both of these differences would contribute to continued catabolism of muscle. The fundamental value to host defense of these systemic metabolic alterations, in particular the increased acute phase globulin synthesis, is further indicated by the fact that they occur in a wide range of inflammatory illnesses and even under circumstances, such as malnourishment, which often lead to reductions in other host defense mechanisms, phagocytosis and cell-mediated immunity.

Possible Involvement of Leukocytic Endogenous Mediator in Granulopoiesis

SummaryRepeated injections of leukocytic endogenous mediator (LEM) were given every 8 hr to rats for 10 days. Peripheral blood neutrophils were elevated rapidly and after several injections were maintained at six- to eightfold normal levels. Mature bone marrow neutrophils were depressed after the first injection and immature marrow neutrophils increased after the tenth injection. Serum from rats receiving multiple. LEM injections had increased amounts of colony-stimulating factor (CSF). Increased bone marrow colony stimulation also resulted when LEM was added directly to agar culture plates of bone marrow cells. This occurred only in the presence of rat serum and the population of adherent bone marrow cells. It was suggested that LEM in the presence of serum acts on mature mono-cytes to promote the release of CSF.The authors thank Mr. Robert McArthur for his excellent technical assistance.

Differentiation between endogenous pyrogen and leukocytic endogenous mediator

Page C15 “Mapes, Carol A., and Philip Z. Sobocinski. Differentiation between endogenous pyrogen and leukocytic endogenous mediator. Am. J. Physiol. 232(1): C15–C22, 1977 or Am. J. Physiol.: Cell Physiol. 1(1): C15–C22, 1977.”

Differentiation between endogenous pyrogen and leukocytic endogenous mediator

The crude material released from glycogen-stimulated rabbit peritoneal polymorphonuclear leukocytes when administered to experimental animals elicits a number of metabolic and physiologic alterations characteristic of those observed in the host inflammatory response. Classically, the mediator of febrile response observed in rabbits and other species has been termed endogenous pyrogen (EP), whereas leukocytic endogenous mediator (LEM) has been used as a general term to denote the substance(s) mediating multiple inflammatory responses observed in rats. The latter substance, however, has not been previously demonstrated to differ from EP. This report presents evidence indicating that EP and LEM are different molecular species. Evidence supporting the differentiation between these entities includes: physical separation of EP from one or more mediators that induce metabolic alterations attributed to LEM; production of LEM activities by stimulated polymorphonuclear leukocytes in the absence of detectable pyrogenic activity; and differences in the release of EP and LEM from stimulated rabbit granulocytes in the presence of potassium ion.