Ralph F. Kampschmidt

Comparison of leukocytic pyrogen and leukocytic endogenous mediator.

Summary Leukocytic pyrogen was subjected to a five-step purification scheme, and quantitative assays for both leukocytic pyrogen and leukocytic endogenous mediator (LEM) were made. At each step of the purification scheme, the active fractions, in addition to producing fever, lowered plasma iron and zinc concentrations, elevated fi-brinogen concentrations, and caused a release of neutrophils from bone marrow. It appears, therefore, that LEM and leukocytic pyrogen are the same molecule.

Effect of Leukocytic Endogenous Mediator on Plasma Fibrinogen and Haptoglobin

Summary A progressive increase in plasma fibrinogen and haptoglobin occurred in rats receiving increasing doses of leuko-cytic endogenous mediator. The increases in fibrinogen occurred sooner after injections of leukocytic endogenous mediator than with endotoxin injections. Neither endo-toxin nor leukocytic endogenous mediator caused increased radioactive glycine incorporation into fibrinogen when incubated directly with the liver in vitro. Both of them when injected into the rat caused a rapid release of a humoral material into the plasma which stimulated in vitro glycine incorporation into fibrinogen.

Neutrophil Releasing Activity in Rats Injected with Endogenous Pyrogen

Summary Partially purified endogenous pyrogen from rabbit polymorphonuclear leukocytes, when injected in rats, will cause neutrophil release from bone marrow. It is suggested that the production of this protein by endotoxin-damaged (activated) leukocytes is responsible for the accelerated release of marrow neutrophils during periods of infection or inflammation.

Effect of Human Monocyte Pyrogen on Plasma Iron, Plasma Zinc, and Blood Neutrophils in Rabbits and Rats

Summary Stimulated 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 Bacteria Endotoxin on Plasma Iron

Summary A single injection of as little as 0.0001 μg of lipopolysaccharide from Escherichia coli produced a hypoferremia in the rat. The maximum decrease in plasma iron occurred 8 to 16 hours after injection. A tolerance to this endotoxin was produced by repeated daily injections. An endogenous, heat labile factor, which would decrease plasma iron, was formed in the plasma, 2 hours after injection of endotoxin.

Effect of Leukocytic Endogenous Mediator on C-Reactive Protein in Rabbits

Summary Elevations in the plasma levels of the acute phase proteins—C-reactive protein (C-RP) and fibrinogen—were found after injection of leukocytic endogenous mediator (LEM) into rabbits. C-RP in the plasma was elevated 8 hr after injection of LEM, and maximum elevation occurred 24 hr after injection. Injection of LEM into rabbits also produced alterations in body temperature, in levels of plasma iron and zinc, and in the number of neutrophils in the peripheral blood.

Effect of extracts from rabbit leukocytes on levels of acute phase globulins in rat serum.

Summary Extracts were prepared from rabbit polymorphonuclear leukocytes by procedures previously shown to yield activity for pyrogenicity and lowering of plasma iron and zinc levels. Rats were injected ip with these extracts, and 10 to 48 hr later their serum was assayed for acute phase globulins by immunological techniques. Positive responses were observed for α1- and α2-AP globulins; whereas responses to injections of control extracts were uniformly negative. The data were interpreted to indicate that one or more proteins synthesized and excreted by leukocytes can act as humoral intermediates in the stimulation of serum acute phase globulins during a variety of acute and chronic disorders.

Possible Involvement of Leukocytic Endogenous Mediator in Granulopoiesis

Summary Repeated 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.

The Effect of Endogenous Pyrogen on the Plasma Zinc Concentration of the Rat

Summary Plasma zinc concentration was depressed in rats following the injection of endogenous pyrogen. The endogenous pyrogen, obtained from peritoneal leukocytes, was active in endotoxin-tolerant animals and repeated injections of this protein did not produce tolerance.

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.