GENERATION OF CHEMOTACTIC ACTIVITY IN RABBIT SERUM BY PLASMINOGEN-STREPTOKINASE MIXTURES

The addition of plasminogen and streptokinase to rabbit serum results in appearance of chemotactic activity which is relatively heat-labile and dialyzable. Generation of this activity requires heat-labile factors in serum, but not the sixth component of complement. The optimal mole ratio for generation of chemotactic activity is 1:1 to 1:3 for plasminogen to streptokinase. Neither material alone is sufficient to generate the activity in fresh serum. Generation of chemotactic activity can be blocked by the presence of ε-aminocaproic acid. This plasmin-generated chemotactic factor sediments slowly in a sucrose density gradient during ultracentrifugation. The addition of plasminogen and streptokinase to rabbit serum containing the preformed chemotactic factor (activated trimolecular complex of C'5, C'6, C'7) causes destruction of the chemotactic factor and loss of hemolytic activity of the sixth component of complement. The same mole ratio of reactants for optimal expression of the phenomenon holds as for plasmin generation of chemotactic activity in untreated serum.     

A PREALBUMIN ACTIVATOR OF PREKALLIKREIN : III. APPEARANCE OF CHEMOTACTIC ACTIVITY FOR HUMAN NEUTROPHILS BY THE CONVERSION OF HUMAN PREKALLIKREIN TO KALLIKREIN

Human plasma kallikrein has been shown to directly and selectively attract human neutrophils from a mixed leukocyte population. The capacity of plasma kallikrein to be chemotactic and to generate the nonapeptide bradykinin was maintained during progressive purification. While neither highly purified prekallikrein nor the prealbumin Hageman factor fragments were chemotactic alone, their interaction so as to convert prekallikrein to kallikrein yielded both chemotactic and kinin-generating activity. Both functions of kallikrein were inhibited by treatment with diisopropyl fluorophosphate, indicating an essential role for the active site of the enzyme in the expression of its chemotactic activity.     

BIOLOGICAL EXPRESSIONS OF LYMPHOCYTE ACTIVATION : II. GENERATION OF A POPULATION OF THYMUS-DERIVED SUPPRESSOR LYMPHOCYTES

A population of thymus-derived lymphocytes has been identified that, upon activation by the nonspecific plant mitogen concanavalin A, suppresses the development of plaque-forming cell responses in fresh or 48-h antigen-stimulated cultures of mouse spleen cells. Suppressor cells can inhibit both primary and secondary IgM and IgG responses in vitro. X-irradiation before activation of peripheral thymus-derived cells by concanavalin A abrogates generation of suppressor cells. After a 48 h activation period, however, the function of concanavalin A-activated suppressor cells is radioresistant. As yet uncertain is whether these suppressor cells are a population of cells distinct from thymus-derived "helper" cells. In certain important regards, the cells mediating these two opposing functions share similar characteristics; the effect observed may be determined by the circumstances of activation or the numbers of activated cells, and may consequently represent different functions of a single thymus-derived regulator cell population.     

Human Plasma Protein C: ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTIVATION BY α-THROMBIN

Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (M(r) = 62,000) contains 23% carbohydrate and is composed of a light chain (M(r) = 21,000) and a heavy chain (M(r) = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human alpha-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity.     

STUDIES ON INFLAMMATION : XVI. ON THE FORMATION OF A CHEMOTACTIC SUBSTANCE BY ENZYMATIC ACTION

Blood serum digested by trypsin yields split products which, when injected into the skin of normal rabbits, rapidly increase the permeability of small cutaneous vessels. This is likewise followed in the affected tissue by prompt local migration of polymorphonuclear leukocytes. The tryptic digest of blood serum can be purified by methods previously described (3). The resulting crystalline substance alters the permeability of the capillary wall and favors local migration of leukocytes in a manner similar to leukotaxine proper. The present observations indicate that leukotaxine, or a substance having at least identical biological properties, appears to have been formed by proteolytic enzymatic action on an otherwise relatively inactive blood serum. This finding serves as additional evidence in support of the view that leukotaxine, recovered from inflammatory exudates, is probably an intermediary product of protein catabolism.     

THE ENCEPHALOMYELITIC ACTIVITY OF MYELIN ISOLATED BY ULTRACENTRIFUGATION

A relatively simple preparation of guinea pig brain myelin, free of gross contamination by other cellular elements has been described. Electron microscopic evidence of the predominance of membranous (lamellar) forms was used as the criterion of purity of this fraction. The slight mitochondrial contamination of the myelin fraction was confirmed by its low succinic dehydrogenase activity. Quantitative bio-assay of the encephalitogenic activity of myelin showed it to have a higher specific activity than whole guinea pig brain. The low encephalomyelitic activity of the other subcellular constituents (nuclei and mitochondria) which were removed from myelin by ultracentrifugation in 30 per cent sucrose could be explained by a small amount of myelin contamination. A basic protein of high specific encephalitogenic activity has been isolated from myelin by methods previously applied to whole brain. Although the protein is similar to nuclear histones, the following facts point to certain significant differences. Nuclei prepared by a different procedure from the one developed for the isolation of myelin were found to be non-encephalitogenic. Although basic protein could be extracted readily from these nuclei by dilute HCl, the same extraction procedure yielded little extractable protein from whole myelin. Myelin which had been defatted by cold chloroform-methanol yielded a basic protein which was highly encephalitogenic. The evidence presented thus supports the view that there exists in myelin a new basic protein responsible for the induction of experimental allergic encephalomyelitis, which is distinctly different from nuclear histones. The possible relationship of this protein to myelin structure and function has been discussed.     

GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO : II. EFFECT OF REPEATED EXPOSURE TO ALLOANTIGENS ON THE CYTOTOXIC ACTIVITY OF LONG-TERM MIXED LEUKOCYTE CULTURES

Mouse cytotoxic T lymphocytes (CTL) were generated in unidirectional mixed leukocyte cultures (MLC) using normal C57BL/6 spleen cells as responding cells and irradiated DBA/2 spleen cells as stimulating cells. Cytotoxicity was assayed on ⁵¹Cr-labeled P-815 (DBA/2) target cells, and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Upon inclusion of 2-mercaptoethanol in the culture medium, it was found that significant CTL activity could be detected for as long as 3 wk in primary MLC. Reexposure of MLC cells to the original stimulating alloantigens after 14–41 days in culture resulted in significant cell proliferation and rapid regeneration of high levels of immunologically specific cytotoxicity. CTL activity in these secondary cultures increased dramatically within the first 24 h and reached higher peak levels than those found at the peak of the primary response. Furthermore, proliferation and reappearance of CTL activity could be demonstrated following each of as many as four sequential alloantigenic stimulations of the same initial cell population at 20-day intervals. Interestingly, cells recovered from MLC at the peak of the primary response on day 4 were insensitive to further allogeneic stimulation. Taken together, these results are consistent with the hypothesis that CTL differentiate in MLC to become long-lived memory cells which gradually lose their cytotoxic activity. Upon reexposure to specific alloantigen, such memory CTL rapidly regain their functional activity and proliferate to generate an expanded CTL population.     

MACROPHAGE-MELANOCYTE HETEROKARYONS : II. THE ACTIVATION OF MACROPHAGE DNA SYNTHESIS. STUDIES WITH INHIBITORS OF RNA SYNTHESIS

Mouse peritoneal macrophages, which do not synthesize DNA in vitro, were fused with melanocytes, a mouse cell strain which proliferates rapidly in vitro. DNA synthesis was induced in macrophage nuclei 2–3 hr after fusion and occurred irrespective of the number of macrophage nuclei present per melanocyte nucleus in each heterokaryon. 50–80% of macrophage nuclei initiated DNA synthesis in the 3–7 hr period after fusion. The activation of most 11–12-day chick red cell nuclei in melanocyte cytoplasm took longer than 10 hr. The lag before DNA synthesis may reflect the heterochromatin content of each nucleus. Studies with actinomycin showed that heterokaryon RNA synthesis was essential for subsequent macrophage DNA synthesis. This RNA was synthesized 1–4 hr before the DNA and was unlikely to be ribosomal RNA, since it was insensitive to <0.1 µg/ml actinomycin. Melanocytes and macrophages were treated before fusion with actinomycin and bromotubercidin to bring about a more selective inhibition of RNA synthesis. Macrophages pretreated for 1 hr with 5 µg/ml of actinomycin showed less than 20% of control RNA synthesis in the first 4 hr after fusion, but a normal activation of macrophage DNA synthesis. Pretreatment of melanocytes for 3–7 hr with 5 µg/ml bromotubercidin, a reversible inhibitor of RNA synthesis, prevented macrophage DNA synthesis without affecting macrophage RNA synthesis in the heterokaryons (81% of control). These studies showed that only melanocyte RNA synthesis was essential for the production of macrophage DNA. The exposure of one cell partner to actinomycin before fusion caused cross-toxicity of the untreated nucleus after fusion. Bromotubercidin, an adenosine analogue which is incorporated into RNA, did not give rise to such cross-toxicity after fusion. Once the macrophage nucleus becomes activated in the heterokaryon it becomes less sensitive to the action of actinomycin.     

BIOCHEMICAL STUDIES ON THE FIBRINOLYTIC ACTIVITY OF HEMOLYTIC STREPTOCOCCI : II. NATURE OF THE REACTION

The fibrinolysin of hemolytic streptococci exerts no hydrolytic action upon casein, gelatin, or peptone. The action on solid human fibrin is characterized by a small and gradual increase in the amino nitrogen content of the solution. The specific and special enzymatic action of fibrinolysin is contrasted with trypsin and with streptococcal peptase (4). Solutions of human fibrinogen, after brief incubation with fibrinolysin, lose the capacity to form fibrin. Solutions of rabbit fibrinogen, on the other hand, retain the property of transformation into fibrin, even after prolonged exposure to fibrinolysin. Qualitative tests, with solutions resulting from the action of streptococcal fibrinolysin on human fibrin, indicate that the end-product may be protein and that the degradation of the molecule is not great.     

STUDY OF TRANSFUSED BLOOD : I. THE PERIODICITY IN ELIMINATIVE ACTIVITY SHOWN BY THE ORGANISM.

Group IV transfused blood in a recipient of unlike group is eliminated by a blood-destroying activity of the body. This blood-destroying activity is periodic both in men and women, and in women coincident with menstruation. The elimination of the transfused blood probably takes place as part of a period of blood-destroying and blood-producing activity of the body, although direct evidence to this effect is so far lacking.     

Eosinopenia of Acute Infection: PRODUCTION OF EOSINOPENIA BY CHEMOTACTIC FACTORS OF ACUTE INFLAMMATION

One distinctive aspect of the response to acute inflammation involves a rapid and persistent decrease in the numbers of circulating eosinophils, yet the mechanisms of this eosinopenia are undefined. One possibility is that the abrupt eosinopenia may be the result of release of small amounts of the chemotactic factors of acute inflammation into the circulation. These studies were designed to examine the numbers of circulating eosinophils after an intravenous injection of zymosan-activated serum, partially purified C5a or the synthetic peptide, N-formyl-methionyl-leucyl-phenylalanine. Each of these factors caused a virtual disappearance of circulating eosinophils within 1 min, a transient return of eosinophils to ~50% of control levels after 10-90 min, and a subsequent decrease which persisted for 5 h. In contrast, the numbers of circulating heterophils, although dropping transiently, rapidly returned and rose to elevated levels for 6 h after injection. The response was not caused by adrenal mediation as it occurred normally in adrenalectomized rabbits. Two chemotaxins of allergic inflammation, histamine and the tetrapeptide valine-glycine-serine-glutamic acid, did not cause significant eosinopenia.     

ENCEPHALOMYELITIS OF MICE : II. A METHOD FOR THE MEASUREMENT OF VIRUS ACTIVITY

In a study of mouse encephalomyelitis the following observations were made:— 1. A definite relationship exists between the amount of virus inoculated intracerebrally and the length of the incubation period. 2. The reciprocal of the incubation period was found to be approximately proportionate to the logarithm of the amount of virus inoculated. The relationship can, therefore, be given a simple mathematical form. 3. This fact provides a basis for a new method for measurement of the activity of mouse encephalomyelitis virus.     

ANTIBODY-MEDIATED ACTIVATION OF A DEFECTIVE β-D-GALACTOSIDASE : II. IMMUNOLOGICAL RELATIONSHIP BETWEEN THE NORMAL AND THE DEFECTIVE ENZYME

Two closely related protein antigens were used to study immunogenic competition. Namely, normal β-D-galactosidase of Escherichia coli (Z) and a genetically defective β-D-galactosidase (AMEF) which seems to differ from the normal in one amino acid substitution. A unique characteristic of this pair of antigens is that, although they are indistinguishable in precipitation and absorption tests with antibodies, the enzymatic activity of AMEF is specifically increased several-hundredfold in the presence of antibodies directed against Z. The following results show that Z and AMEF also differ in their immunogenic ability: (a) antibodies directed against Z activated AMEF; antibodies directed against AMEF did not activate, but competed specifically with activating antibodies. (b) Animals immunized with AMEF failed to produce activating antibodies when they were subsequently challenged with Z, although the presence of some cells primed to produce activating antibodies could be demonstrated by adoptive transfer. (c) Animals preimmunized with Z were stimulated in their production of activating antibodies by AMEF challenge, although not as efficiently as with Z. A model explaining these observations by competition for the immunogenic site among antigen-sensitive cells carrying cross-reacting receptors is presented.     

OBSERVATIONS ON THE PHAGOCYTOSIS OF THE PNEUMOCOCCUS BY HUMAN WHOLE BLOOD : II. THE NEUTRALIZATION OF THE ANTI-PHAGOCYTIC ACTION OF THE SPECIFIC SOLUBLE SUBSTANCE BY ANTISERUM

1. In vitro phagocytic experiments with human blood, antipneumococcus serum, pneumococcus specific soluble substance, and living virulent pneumococci show that there is a definite phagocytic inhibition zone when strong antiserum is used. 2. If the antiserum is further diluted, there is a zone where phagocytosis is effective. If the serum is diluted still more, phagocytosis gradually falls off, as the very dilute antiserum fails to neutralize the specific carbohydrate, which has a specific antiphagocytic action. 3. The inhibition zone is apparently caused by the specific precipitate (formed by the antiserum and the specific carbohydrate) interfering, perhaps mechanically, with the ingestion of the pneumococci by the leucocytes. 4. The inhibition zone is better marked with Type III than with Type I pneumococcus. 5. As the concentration of antiserum in the zone of effective phagocytosis in vitro does not correspond with the concentration of antiserum generally used in vivo in the serum therapy of pneumonia, this question is discussed.     

STUDIES ON OXIDATION AND REDUCTION BY PNEUMOCOCCUS : II. THE PRODUCTION OF PEROXIDE BY STERILE EXTRACTS OF PNEUMOCOCCUS.

In the present paper methods have been described for the preparation of sterile extracts of pneumococci. These extracts may be obtained by dissolving the bacteria in broth cultures by means of bile, or by extraction of the cellular substances by repeated freezing and thawing of broth or saline suspensions of unwashed cells. Under special precautions these extracts may be passed through Berkefeld filters without loss of potency. In this procedure, as in all other manipulations incident to their preparation, the extracts should be protected as far as possible from contact with air. All extracts were proved sterile by cultural and animal tests. Sterile extracts of unwashed pneumococcus cells promptly form peroxide on exposure to air. Peroxide formation is almost as active in extracts aerated at 2°C. as in those exposed to the air at room temperature. Detectable amounts of peroxide may be produced by these cell extracts within the reaction range of pH 5 to 9, the optimal zone lying at reactions less acid than pH 6. The peroxide-forming activity of the extracts is gradually diminished by prolonged exposure to 55°C., and is completely destroyed by heating at 65°C. for 5 minutes. Cell extracts of pneumococci which have been thoroughly washed prior to extraction in salt or phosphate solutions exhibit no peroxide-forming activity. These extracts of washed cells may be activated by the addition of the cell washings, yeast extract, or muscle infusion.     

STUDIES ON SPREADING FACTORS : II. THE EFFECT OF SERUM UPON HYALURONIDASE SPREADING ACTIVITY

The reaction between normal serum and hyaluronidase has been studied in vitro and under in vivo conditions in skin. Using in vitro conditions of incubation, serum exhibits antihyaluronidase activity as measured by assay of hyaluronidase spreading activity in skin. This confirms the work of others, who have previously described the serum inhibitory factor using other tests of hyaluronidase activity. When, however, hyaluronidase and setum are allowed to incubate in skin under in vivo conditions, no inhibitory influence of serum upon hyaluronidase spreading activity is evident. This latter finding has been taken to indicate that the environmental conditions in skin are unfavorable for the inhibitory reaction of serum upon hyaluronidase. The disparity between the in vivo and in vitro effectiveness of serum, and the significance of the serum factor as a defense mechanism against invasive processes, have been briefly discussed.     

IN ACTIVATION OF B. SUBTILIS SPORES AND E. COLI ENDOTOXIN BY ETHYLENE OXIDE

Exposure of Bacillus subtilis spores to ethylene oxide (EO) showed correlation between the killing rate and the EO concentration, when the temperature was kept at 55 degrees C and the relative humidity at 100%. The co-efficient of dilution was calculated to be 0.9. The effect of EO on Escherichia coli endotoxin was investigated by the chromogenic Limulus Amebocyte Lysate (LAL) test. A solution of endotoxin was dried on glass tubes and exposed to 450 or 900 mg EO/l during 1-46 h under the same conditions as the spore inactivation. The LAL activity of the endotoxin was reduced to about 30%. The EO-treated endotoxin was tested in the rabbit pyrogen test. The summed temperature increase for three rabbits was 0.9 degrees C, while the same assay using untreated test pieces showed an increment of 3.7 degrees C. Administration of the same quantity of EO-treated and untreated endotoxin to the rabbits, as adjusted by the LAL-test, produced the same temperature increment. The addition of polymyxin B (PB) to an endotoxin solution reduced the LAL activity by 75%. Had the endotoxin been exposed to EO, thereby reducing the LAL activity by 70%, addition of PB further reduced the activity by 99%. The reaction of EO on the endotoxin reduced the LAL activity as well as the pyrogenic response and increased the affinity to PB.