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Interactions between rabbit polymorphonuclear leucocytes and staphylococci.

A method has been described for the study in vitro of leucocyte-bacteria interactions which permits the simultaneous evaluation of both phagocytosis and intracellular bacterial inactivation. Employing this technique, the fate and localization of staphylococci in homogeneous suspensions of rabbit polymorphonuclear leucocytes have been studied. Coagulase-positive strains of S. aureus were not efficiently ingested in the presence of normal rabbit serum. In contrast, coagulase-negative strains of S. albus were rapidly engulfed and inactivated. Immune sera prepared against a coagulase-positive strain enhanced the the ingestion of the homologous organism as well as of three heterologous strains of S. aureus. Following phagocytosis, prompt intracellular killing of S. aureus occurred. The thermostable opsonins in immune sera reacted only with strains of S. aureus. A comparison between polymorphonuclear leucocytes obtained from normal and immune animals revealed no differences in their ability either to ingest or kill coagulase-positive staphylococci. Studies with other bacterial species are presented to illustrate: (a) phagocytosis followed by intracellular inactivation; (b) phagocytosis followed by intracellular survival; and (c) the absence of phagocytosis.

Animals↗

The role of polymorphonuclear leukocytes in the initiation and cessation of the Arthus vasculitis.

The role of polymorphs in the Arthus type hypersensitivity vasculitis has been studied. Polymorphs were found to play an essential role in not only producing the inflammatory vasculitis, but also were instrumental in ridding the damaged vessel of the antigen, probably by means of proteolytic catabolism at the inflammatory site. A temporal relationship between the disappearance of antigen from the damaged vessels and a decrease in inflammatory reaction was found. The earliest localization of antigen and its associated rabbit globulin in the Arthus vasculitis was found to be beneath the endothelium of small vessels. Since submitting this article for publication, Sorkin and Boyden (J. Immunol., 1959, 82, 332) have reported the catabolism of antigen in the presence of antibody by mononuclear cells obtained from the peritoneal cavities of guinea pigs. Evidence was presented indicating that the antigen molecule was actually broken down by the mononuclear cells. Techniques similar to those reported here were used.

Animals↗

Studies on the pathogenesis of fever. VI. The interaction of leucocytes and endotoxin in vitro.

Study in vitro of the interaction of bacterial endotoxin with rabbit polymorphonuclear leucocytes has resulted in the following findings: 1. Incubation of endotoxin and leucocytes in saline results in: (a) the release of leucocytic pyrogen, and (b) the inactivation of endotoxin. 2. Cell-free extracts of leucocytes also inactivate endotoxin. 3. Incubation of leucocytes in "physiological" saline causes rapid discharge of leucocytic pyrogen. In contrast, relatively little pyrogen is released by leucocytes incubated in fresh serum. 4. The release of leucocytic pyrogen in serum is markedly stimulated by the presence of endotoxin. 5. Leucocytes obtained from tolerant rabbits interact with endotoxin in essentially the same manner as leucocytes from normal rabbits. The pertinence of these findings to the pathogenesis of fever and to related information concerning human leucocytes has been discussed.

Animals↗

Studies on the pathogenesis of acute inflammation. II. The relationship of fibrinogen and fibrin to the leucocytic sticking reaction in ear chambers of rabbits injured by heat.

The relation of intravascular fibrin to the leucocytic sticking reaction in ear chambers of rabbits injured by heat was investigated in two ways. First, attempts were made to destroy the thin layer of fibrin believed to coat the surfaces of cells involved in the sticking reaction. Second, white cell sticking was studied after fibrinogen had been removed from the blood stream. The results of these experiments were as follows:- 1. Activation of fibrinolysin in vivo by streptokinase did not impair sticking of white blood cells. 2. Administration of streptokinase parenterally did not lower fibrinogen blood levels appreciably even when the amount used was large. 3. Thromboplastin infusions alone reduced circulating fibrinogen to low levels but leucocytic sticking was not prevented. Furthermore, frequent death of animals due to pulmonary embolism made such experiments prohibitive. 4. Addition of streptokinase to thromboplastin infusions protected against embolic deaths but did not influence sticking even though the fibrinogen levels achieved were quite low. 5. Finally, when thrombin was added to infusions of thromboplastin and streptokinase, no circulating fibrinogen could be detected. Under such circumstances leucocytic sticking following heat injury occurred without reduction. These findings were interpreted as evidence against a primary role of the blood clotting mechanism in causing the sticking of white blood cells to injured endothelium. Alternative explanations were discussed.

Animals↗

Observations on the production of pyrogenic substances by rabbit and human leucocytes.

1. The mechanism of release of a pyrogen from leucocytes has been studied in cells obtained from sterile rabbit peritoneal exudates and from rabbit blood. Attempts were made to induce human leucocytes-from blood-to release a pyrogen. 2. Rabbit leucocytes, kept below 4 degrees C., were not pyrogenic and did not release any pyrogen when disintegrated. Incubating such cells, in various media, at 37 degrees C. led to the formation of a pyrogen which was heat-labile. The maximum yield was attained after 1(1/2) hours' incubation. 3. The formation of rabbit leucocytic pyrogen was prevented by freezing and thawing the leucocytes, by heating them to 56 degrees C. for half an hour before incubation, and by ageing them in the cold. 4. Nitrofurazone (5-nitro-2-furaldehyde semicarbazone) prevents the formation of leucocytic pyrogen when given by mouth to the cell-donor animals, or when added to leucocytes in intro. 5. Leucocytes from rabbit blood formed leucocytic pyrogen, on incubation in saline, and this formation was also inhibited by nitrofurazone. 6. No leucocytic pyrogen was released from human leucocytes subjected to mechanical, osmotic, or thermal damage, and it was not formed when the cells were incubated in saline. 7. The source of rabbit leucocytic pyrogen, the action of nitrofurazone on leucocytes, and the supposed role of leucocytic pyrogen in fever are discussed.

Animals↗

Factors affecting the adhesiveness of human leucocytes and platelets in vitro.

Some factors affecting the retention of human polymorphonuclear neutrophils (PMN), lymphocytes, and platelets on a siliconized glass bead column were explored. PMN were more effectively retained when the flow rates were slow and the columns long. They were found largely on the upper portions of the columns except with rapid flow rates when they spread down the columns. PMN retention on the columns was greatest in the range 30 degrees -43 degrees C. Both magnesium and calcium ions were required for full adhesiveness; calcium ions alone were unable to restore adhesiveness to PMN from blood which had been treated with a chelating resin to remove divalent cations. The adhesiveness of the PMN was independent of cyanide and dinitrophenol, but was almost completely eliminated by iodoacetamide. Under all the conditions mentioned above in which adhesiveness was lost there was a concurrent loss of the usual ability of the PMN to migrate, but at least in the presence of EDTA, a capacity to change shape by pseudopod formation remained. Lymphocytes were retained on the columns to a much lesser extent than the PMN under all conditions and, within limits, this retention was not related to either flow rate or column length. Maximum lymphocyte retention occurred in the range 30 degrees -43 degrees C. No dependence of lymphocyte adhesiveness was shown for divalent cations, cyanide, dinitrophenol, or iodoacetamide, but such dependence is not excluded by the data obtained. Platelets were largely retained by the glass bead columns under most conditions and this was unrelated to temperature in the range 0 degrees -50 degrees C. Their adhesiveness was found to require either magnesium or calcium ions and to be blocked by iodoacetamide.

Adhesiveness↗

The egestion of phagocytized particles by leukocytes.

Streptococci that were phagocytized by human or mouse leukocytes were usually enclosed in a digestive vacuole. At times the vacuole emptied itself, discharging the cocci to the outside. Whether the egested streptococci then multiplied in the extracellular environment appeared to depend upon how long they had been in the vacuole. The phenomenon of egestion also involves particles other than bacteria, as shown by observation of an erythrocyte that was phagocytized by a human leukocyte and was hemolyzed intracellularly, forming a cell ghost that was then egested.

Animals↗