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Electron microscopic observations of rabbit leucocytic pyrogen
EXPERIMENTAL
AND MOLECULAR
Electron
3,
PATHOLOGY
19-30
Microscopic
(1964)
Observations
Leucocytic K. Department
E. FRITZ,
of
Pathology,
G.117.
Albatty Received
of
Rabbit
Pyrogen’ GANDER,
Medical October
AND
F.
College,
GOODALE?
Albany,
New
York
31, 1962
INTRODUCTION Hit look lak sparrer-grass, Hit tas’e lak sparrer-grass,
hit feel lak sparrer-grass, en I bless ef ‘taint sparrer-grass. *‘Nights
with Uncle Remus,” 1883 Joel Chandler Harris
Studies on the pathogenesis of fever have identified certain pyrogenic substances (Menkin, 1943; Wagner et al., 1949; Bennett and Beeson, 1950; Snell, 1962). Among these is the pyrogen liberated by rabbit or human leucocytes that have been artificially stimulated by contact with saline or with bacterial endotoxin (Beeson, 1948; Cranston et al., 1956). The leucocytic pyrogen of both rabbit and man is a nondialyzable protein which is heat labile (pyrogenicity destroyed by 5 minutes at 6O”C), does not produce tolerance upon repeated injection, and is characterized by a relatively short latent period between intravenous injection and the onset of fever (Snell et al., 1956; Rafter et al., 1960; Gander and Goodale, 1962a, b). These properties are in contrast to those of bacterial pyrogens which are heat stable, produce tolerance after repeated injection, and have a significantly longer latent period before the onset of fever. Individuals tolerant to bacterial endotoxin show no diminution in their pyrogenic responseto leucocytic pyrogen. The processby which leucocytes produce their pyrogen is not yet known. Examination by light microscopeof leucocytes that have been artificially stimulated to release their pyrogen reveals no recognizable alterations from normal leucocytes. With electron microscopy, however, rabbit and human leucocyte preparations so stimulated show a granular material in close juxtaposition to normal appearing leucocytes (Goodale et al., 1962a) (Figs. 1 and 2). 1Ve believe that this material represents, at least in part, leucocytic pyrogen. A morphologically identical granular material appears in and around leucocytes from human patients who were naturally febrile from a variety of diseaseprocesses(Goodale et al., 1962b) (Fig. 3). Positive identification of the granular substance as leucocytic pyrogen has not yet 1 This work was supported by U. S. Public Health Service Grant E-3720, National Institute of Allergy and Infectious Diseases. Presented in part at the Fifth International Congress for Electron Microscopy, Philadelphia, Pa., August 29-September 5, 1962. :! Present address: Department of Pathology, iMedical College of Virginia, Richmond, Virginia. 19
so
FIG. Lilxxal).
K.
1.
13. FRITZ.
G. W.
GANDKH,
.AKl)
17. GO01~A1,li
Rabbit Irucoq~te aitel- incubation it;! I huur \qith Iwtcrial cndutuxin (O..j!Lg oi Above is part of a platelet ; below. part oi the leucoqtr cytoplam and nucleus. Rctween them is a 100x agarcgate of line granules. Apparent defects in Icucocyte plasma membrane, such as seen here: are not necessary related to granular aogrepates. Rounded bodies in and around agpregate are probably cross section5 of cytoplasmic processes. Unstained. X 50.000.
FIG. .3. Portion of leucocyte from a patient febrile with urinary tract infection. So incubation with bacterial endotoxin. Aggregates of granular material apparmtly within cell vacuoles ma) have been phagocytized, may have been formed within the vacuole, or may, because of the plane 01 section, actually be outside the cell. Appearance of granular material here is similar lo that in rabbit and human leucocpte preparations artificially stimulated i11 aitro with bacterial endotoxin (Figs. 1 and 2). Size of individual granules formin p argreaatcs _ in FiKs. l-3 measures from SO to loo.&, although it is obvious that these mcasuremcnts can only be approximate in such a preparation and at such a magnification. Unstained. X 50,000.
RABBIT
LEUCOCYTIC
23
PYROGEN
been made, but confirmatory evidence is accumulating. Some of this evidence has come from the study of chemically purified rabbit leucocytic pyrogen by electron microscopy. This report describes the electron microscopic appearance of purified rabbit leucocytic pyrogen, and compares it morphologically with crude rabbit leucocytic pyrogen. We have examined rabbit pyrogen initially in preference to human pyrogen because it is easier to obtain in quantity and therefore in more pure form. Also, preliminary studies in this laboratory indicate that human and rabbit leucocytic pyrogen are chemically similar (Gander and Goodale, 1962b). MATERIALS
AND
LEUCOCYTE
METHODS
PREPARATIONS
Leucocytes seen in Figs. l-3 were obtained and prepared as previously (Goodale et al.. 1962a). PURIFICATION
OF RABBIT
LEUCOCYTIC
described
PYROGEN
The materials and methods used in partially purifying rabbit leucocytic pyrogen have been described elsewhere in detail (Gander and Goodale, 1962a). Briefly, the crude pyrogen, obtained from the leucocytes of sterile peritoneal exudates, is dialyzed against distilled water, freeze-dried, and applied in phosphate buffer first to a DEAE (diethylaminoethyl) cellulose column, which removes most of the protein, and then to a phosphorylated cellulose column which adsorbs leucocytic pyrogen while allowing virtually all of the remaining protein to be eluted. The pyrogen, in turn, is eluted by a stepwise increase in pH with a resulting eightyfold increase in pyrogenic activity per gram of protein as compared with the crude pyrogen. This pyrogenic protein is inactivated by heating at 60°C for 5 minutes or by trypsin digestion. It is electrophoretically homogeneous at pH’s 5.0 and 8.0. PREPARATION
Direct application
OF
PURIFIED
PYROCEN
FOR
ELECTRON
MICROSCOPY
of pyogen to grid
.d. The first method used was to transfer a very small drop of the pyrogenic protein solution to a Formvar-coated grid by means of a capillary tube, and to allow it to dry. The grid was then exposed to osmium tetroxide vapor for 15 minutes. B. Later, an adaptation of a method described by Porter was used and gave much better results (Porter and Hawn, 1949). Glass slides were cleaned twice in 95% ethanol, dried each time with a lint-free cloth, immersed momentarily in a recently filtered solution of 0.15% Formvar in ethylene dichloride, removed, and allowed to drain and dry in a dust-free area. Using a capillary tube, a thin film of the pyrogenic protein solution was spread over the surface of the Formvar, any excess drained, and the glass slide exposed while wet to osmium tetroxide vapor in a water saturated atmosphere in a covered Petri dish for 15 minutes. The Formvar-coated slide was then removed, the surface rinsed with distilled water, and with a sharp needle the Formvar film was cut into segments approximately 2 mm square. The squares were then floated off the slide onto the surface of distilled water and picked up on acetonecleaned grids raised from below the surface of the water. After drying at room temperature the grids were examined with the electron microscope.
24
K.E.FRITZ,G.
W.GANDER, AND F.GOODALE
C. The pyrogenic protein was also examined after preparation by the negative staining method of Brenner and Horne (1959). A drop of a solution composedof equal parts of 2% aqueous phosphotungstate solution (pH 7.4) and a solution of the pyrogenic protein in phosphate buffer was applied directly to Formvar-coated grids. After drying, the grids were ready for examination, although in someinstances the grids were exposed to osmium tetroxide vapor for 15 minutes prior to examination. D. Protein solution was applied to Formvar-coated grids, air dried, then either exposed to osmium tetroxide vapor for 15 minutes or stained with 0.1% phosphotungstic acid, and shadowed with chromium by an adaptation of Hall’s method to available equipment (Hall, 1949). THIN-SECTION
PREPARATIONS
OF PYROGEN
To prepare samples of pyrogenic protein for thin sectioning, a solution of the protein was first evaporated to dryness in a small glasstube, leaving a minute amount of barely visible, soft, white powdery residue adhering to the walls of the tube. Prepolymerized methacrylate (7 parts butyl methacrylate to 1 part methyl methacrylate) with catalyst ( 1.5% benzoyl peroxide) was introduced into the tube, using great care not to agitate the powder, and the methacrylate hardened at 50°C for 1 week. Following this, the glasstube was broken carefully, pieces of glasswere peeled away from the methacrylate mass, and the methacrylate then examined by phase microscopy. Wherever inclusions of the powdery residue in the methacrylate were found they were cut out and re-embedded in methacrylate in gelatin capsules. Following polymerization, thin sections were cut with glass knives on a Servall Porter-Blum microtome, mounted on Formvar-coated grids, and exposed to osmium tetroxide vapor for 15 minutes. CONTROLS
Protein-free buffer solutions of the type used in the purification of the protein were passedthrough both cellulosecolumns and prepared for the electron microscope as described above in Part B under Direct application of pyrogen to grid. Thin sections of buffer were also prepared as described under Thin-section preparations of pyrogen. All grids were examined with a SiemensElmiskop I electron microscope operated at 60 KV at instrumental magnifications up to 40,000 X using a double condenser lens system with a beam diameter of 5 u. RESULTS DIRECT APPLICATION
OF PYROGEN TO GRID
.4. When the solution containing pyrogen was transferred directly to a Formvarcoated grid, allowed to dry, and then exposed to osmium vapor, examination revealed areas of two types. One contained large, dense,black amorphousprecipitates in which no detail could be discerned. The other showed less dense,finely granular precipitate which bore a striking resemblanceto the granular material seen in pyrogenic white cell preparations. It was difficult, however, to distinguish by this method morphological characteristics of the individual particles which composethe precipitate.
H. In contrast. micrographs of preparations, made by subjecting a thin wet ftlm of Ixotein solution on a Formvar-coated grid to osmium vapor in a water-saturated atmosphere. showed the protein to be made up of discrete. densely osmiophilic dots or particles of about 2.5 A diameter (Fig. 4). They were separated II); a relativel!- con-
stant minimum distance, the distance from dot center to dot center being appro.ximately 50 A. The area between the densely osmiophilic dots was faintly osmiophilic when compared with the background. Excellent dispersion was achieved by this preparative method probably because only the fixed particles directly adjacent to the Formvar film adhered to the lilm through the washing process. Thus the layer ot adherent particles approached a monolayer and sul,eriml,ositioll was minimized. Measurement of the particles was probably more nearly representative of their actual dimensions than would be true if the protein film were allowed to dry before being exposed to osmium vapor. Stresses of dryin: tend to ilatten particles into the supand decreasing their vertical dimensions. portin, 1~ film. thus increasing their horizontal To some extent. at least, it is likely that the iisation of the particles while in solution helps them to withstand dryin, (J with less distortion. (‘. Electron micrographs of the negatively stained protein revealed areas in which the dense black phosl,hotunestate background outlined discrete: unstained spheres (Fig. j) averaging approximately 50 ;i in diameter. So dense, black dots appeared of negatively stained protein in this type of preparation. However. in micrographs
prepared in exactly the same way but then subjected to osmium tetroside vapor: dense, black dots now appeared in the center of the unstained spheres (Fig. 6). Measurements of the dots and of the unstained areas corresponded closely to the measurements of the dots and spacing as shown in the other preparations. Negative staining, however, also brought out the fact that the spheres with their central dots tended to occur in clusters or aggregates of 20-30.
YlG. 5. Pyrogen-containing rxposure to osmium vapor. unstained spheres of about
negatively stained with phosphotunjistic solution JIknse T-shaped hack~round of phosphotun&ic acid 50 i\ diameter.
acid, without outlines indkidual
II. Examination of shadowed preparations revealed some spherical particles. the smallest of which measured about 45 A in diameter. However, the grain size of the deposited chromium metal was so large that the protein particles did not appear in sufficient contrast to warrant conclusions being drawn as to their structure or dimensions. With the availability of more versatile vacuum evaporating equipment, information of considerable import should be obtainable from shadowed preparations.
Micrographs of thin-sectioned preparations of the pyrogen (Fig. i ) again showed densely osmiophilic particles of about 25 A diameter, separated by a relatively constant distance of 50a from particle center to particle center. This appearance is thus the same as that demonstrated when the protein is not thin-sectioned but applied directl!: to the grid surface.
FIN:. 6. Preparation idenlical to that seen in FiC. 5. and thrn exposed 10 osmium u~lor. 111 the center of the .50 .k spheres a densely osmiophilic dot of about 35 A diameter has appeared. .is in Fig. 5 the 50 .% spheres tend lo appear in TWUJX ~ Of d,OUt .
FIG. 7. A thin section Section exposed to fxmium
of methac~);late-cmI,edded vapor. .+T-era;r particle
r&due from p)ru~en-contaillin~ size is about 25 A.
solution.
28
K. E.FRITZ>
G. W. GANDER, CONTROL
AND
F.GOODALE
PREPARATIONS
Micrographs of protein-free buffer solutions that had been passed through both cellulose columns, then thin-sectioned or applied directly to Formvar-coated grids and exposed to osmium tetroxide vapor, showed occasional crystals, but nothing resembling the densely osmiophilic particles of the preparations containing pyrogen. DISCUSSION Both crude and purified leucocytic pyrogen, when stained only with osmium, appear under the electron microscope as particles of two sizes: the smaller and more numerous measureabout 25 A and the larger, 700-800 ii in diameter. In both pyrogens the distance from each small particle center to the center of its neighbor is remarkably constant at about 50 A. The particle size by these measurementswas puzzling becausewhen we calculated the molecular weight for particles of 25 A diameter (assuming a density of 1.3 for dry, unfixed protein) we arrived at a figure of 6000. Because the pyrogenic protein is nondialyzable, such a molecular weight would be quite unreasonable. However, when the pyrogen was negatively stained, its basic structural unit was clearly not a 25 ii particle but a sphere 50 A in diameter in the center of which, when osmium staining was added to the negative staining, a densely osmiophilic core of 25 A diameter appeared. Now we could explain why the smallest unit or particle of the crude osmiumstaIned pyrogen (for which we do not yet have a negative staining technique) appears to be 25 ii in diameter and also why there exists the constant distance of 50 A separating these small particles. In addition, a basic unit of 50 A diameter would have a molecular weight of about 50,000, which is reasonable in view of our knowledge of the chemical properties of the pyrogen. Negative staining of the purified pyrogen also re-emphasizesthat the 50 A spheres tend to form aggregatesof 600-800 A in diameter. In a morphologic study such as this, interpretation of results must be made with extreme caution, perhaps even skepticism. Before assuming that we have indeed visualized moleculesof leucocytic pyrogen we must consider these possiblesourcesof error: I. The granular material seen in leucocyte preparations (which contain crude pyrogen) could be preparative artifact or cell product other than leucocytic pyrogen. However, in a previous publication we have stated our reasons for believing that the particles do, in fact, represent crude leucocytic pyrogen (Goodale et al., 1962a). 2. The particles we have visualized in preparations of purified pyrogen may not be pyrogen but another protein (or proteins) so similar in chemical and physical properties as to be inseparable from the pyrogen by our present purification procedure. This is possible even though the purified protein is electrophoretically homogenous at several different pH values. 3. The particles which we have shown in purified pyrogen preparations might represent a contaminant acquired during purification. The best evidence against this possibility is that buffer solution subjected to the samepurification processdoes not show the osmiophilic particles characteristic of the pyrogenic fractions. Added evidence against their being a contaminant is suggestedby the absenceof the characteristic particles in the nonpyrogenic fractions of the column eluate so far examined.
RABBIT
LEUCOCYTIC
PYROGEN
29
4. Perhaps the particles we are visualizing are a result of preparing the protein for electron microscopy rather than being the size and shape of the protein itself. The most cogent reason for believing that this is not so is that, regardless of the method of preparation (be it thin film in osmium tetroxide vapor, thin section, or negatively stained), the resultant particles are remarkably consistent in appearance and dimensions. 5. The spheres (if we assume 3 dimensions) of 50 j, diameter that we have demonstrated may not be molecules but instead either agglomerates of smaller molecules or fragments of larger molecules. These possibilities cannot be ruled out at present. 6. Morphological similarities between particles seen in pyrogenic cell preparations and those seen in preparations of purified pyrogen obviously do not mean that the two are identical. At best the evidence linking them is circumstantial, and rests on the fact that they appear in pyrogenic preparations and not in nonpyrogenic ones. After considering these possible misinterpretations of our electron micrographs we believe that we have visualized a protein (or proteins) which is either leucocytic pyrogen or protein thus far indistinguishable from it. The particle size is such that it might reasonably represent the intact pyrogen protein molecule. Some evidence suggests that the particulate substance in pyrogenic cell preparations may be the same substance visualized in preparations of purified pyrogen. Assuming that we have visualized the pyrogen molecule. two points of interest arise. First, the molecular weight of 50,000 calculated from the size of the molecules is helpful in our study of a substance which is available in such small quantities. It also seems reasonable in the light of what we know of the properties of the pyrogen. The second point of interest is the chemical and biological significance, if any, of the densely osmiophilic core. The possibility that the site of pyrogenicity may be associated with it is intriguing. Also it may represent a structural concentration of certain amino acids. Of the seven amino acids thus far tentatively identified in the pyrogen. three (tryptophan, arginine, and histidine) are known to react strongly with osmium tetroxide, at least in the test tube (Bahr, 1954). The most feasible approach to positively identifying the visualized protein as the pyrogen is immunologic. If we can successfully produce specific antibodies to the pyrogen, as we are now attempting to do. we will have available a tool of greatest potential for our study. The possible labeling of such antibodies with electron dense substances such as ferritin or uranium should help us localize and identify the pyrogen by electron microscopy in conjunction with immunochemical studies. SUMMARY We have studied by electron microscopy a pyrogenic protein derived from rabbit leucocytes. Whether the pyrogen is in crude or purified form. the basic unit appears the same: a sphere jo A in diameter which has a central, densely osmiophilic core of about 25 A diameter. The reasons for believing that these particles may represent the substance we know chemically as leucocytic pyrogen are: (1) they are consistently present in preparations known by biological or chemical assay to contain leucocytic pyrogen; they are consistently absent in nonpyrogenic controls, (2) The calculated molecular weight of the particulate material (50,000) is consistent with our knowledge of the chemical properties of the pyrogen. (3) We cannot find any preparative artifact or other substance that this particulate material might reasonably repesent. Quite obviously none of our 3 reasons constitutes proof that what we are seeing is really the pyrogen. Although it seems probable to us that we are visualizing the pyrogen, we must wait until we develop an antibody to the pyrogen before we can present more convincing evidence,
30
K.
E.
FRITZ,
G. W.
GANDER,
AND
F.
GOODALE
REFERENCES BAHR, G. F. (1954). Osmium tetroxide and ruthenium tetroxide and their reactions with biologically active substances. Exptl. Cell Res. 7, 459-479. BEESON, P. B. (1948). Temperature-elevating effect of a substance obtained from polymorphonuclear leucocytes. 1. Clin. Invest. 27, 524. RENNET, I. L., JR., and BEESON, P. B. (1950). The properties and biologic effects of bacterial pyrogens. Med. 29, 365-400. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. Biochem. Biophys. Acta 34, 103-110. CRANSTON, W. I., GOODALE, F., SNELL, E. J., and WENDT, J. F. (1956). The role of leucocytes in the initial action of bacterial pyrogens in man. Clin. Sci. 16, 2 19-226. GANDER, G. W., and GOODALE, F. (1962a). Chemical properties of ieucocytic pyrogen. I. Partial purification of rabbit leucocytic pyrogen. Exptl. Mol. Puthol. 1, 417-426. GANDER, G. W., and GOODALE, F. (1962b). A method for purification of human leucocytic pyrogen. Proceedings of the Ninth Congress of the International Society of Hematology, Mexico City, September (Abstract). GOODALE, F., FILLMORE, R., and HILLMAN, E. (1962a). Electron microscopic observations of human leucocytes. I. Response in vitro to bacterial endotoxin. Ewptl. Mol. Pathol. 1, 229-250. GOODALE, F., HILLMAN, E., and FILLMORE, R. (1962b). Observations of human leucocytes from patients with naturally occurring fevers. In “Proceedings of the Fifth International Ccngress for Electron Microscopy” (S.S. Breese, ed.), Vol. 2, p. SS-3. Academic Press, New York. HALL, C. E. (1949). Electron microscopy of fibrinogen and fibrin. J. Biol. Chem. 179, 857-864. MENKIN, V. (1943). Chemical basis of injury in inflammation. Arch. Pathol. 36, 269-288. PORTER, K. R., and HAWN, C. Z. (1949). Sequences in the formation of clots from purified bovine fibrinogen and thrombin: A study with the electron microscope. J. Exptl. Med. 90, 225-232. RAFTER, W. S., COLLINS, R. D., and WOOD, W. B., JR. (1960). Studies in the pathogenesis of fever. VII. Preliminary chemical characterization of leucocytic pyrogen. J. Exptl. Med. 111, 831-840. SNELL, E. S., GOODALE, F., WENDT, F., and CRANSTON, W. I. (1956). Properties of human endogenous pyrogen. Clin. Sci. 16, 615-626. SNELL, E. S. (1962). Pyrogenic properties of human pathological fluids. CZin. Sci. 23, 141.150. WAGNER, R. R., BENNETT, I. L., JR., and LEQUIRE, V. S. (1949). The production of fever by influenza1 viruses. I. Factors influencing the febrile response to single injections of virus. Z. ExjtZ. Med. 90, 321-334.
AND MOLECULAR
Electron
3,
PATHOLOGY
19-30
Microscopic
(1964)
Observations
Leucocytic K. Department
E. FRITZ,
of
Pathology,
G.117.
Albatty Received
of
Rabbit
Pyrogen’ GANDER,
Medical October
AND
F.
College,
GOODALE?
Albany,
New
York
31, 1962
INTRODUCTION Hit look lak sparrer-grass, Hit tas’e lak sparrer-grass,
hit feel lak sparrer-grass, en I bless ef ‘taint sparrer-grass. *‘Nights
with Uncle Remus,” 1883 Joel Chandler Harris
Studies on the pathogenesis of fever have identified certain pyrogenic substances (Menkin, 1943; Wagner et al., 1949; Bennett and Beeson, 1950; Snell, 1962). Among these is the pyrogen liberated by rabbit or human leucocytes that have been artificially stimulated by contact with saline or with bacterial endotoxin (Beeson, 1948; Cranston et al., 1956). The leucocytic pyrogen of both rabbit and man is a nondialyzable protein which is heat labile (pyrogenicity destroyed by 5 minutes at 6O”C), does not produce tolerance upon repeated injection, and is characterized by a relatively short latent period between intravenous injection and the onset of fever (Snell et al., 1956; Rafter et al., 1960; Gander and Goodale, 1962a, b). These properties are in contrast to those of bacterial pyrogens which are heat stable, produce tolerance after repeated injection, and have a significantly longer latent period before the onset of fever. Individuals tolerant to bacterial endotoxin show no diminution in their pyrogenic responseto leucocytic pyrogen. The processby which leucocytes produce their pyrogen is not yet known. Examination by light microscopeof leucocytes that have been artificially stimulated to release their pyrogen reveals no recognizable alterations from normal leucocytes. With electron microscopy, however, rabbit and human leucocyte preparations so stimulated show a granular material in close juxtaposition to normal appearing leucocytes (Goodale et al., 1962a) (Figs. 1 and 2). 1Ve believe that this material represents, at least in part, leucocytic pyrogen. A morphologically identical granular material appears in and around leucocytes from human patients who were naturally febrile from a variety of diseaseprocesses(Goodale et al., 1962b) (Fig. 3). Positive identification of the granular substance as leucocytic pyrogen has not yet 1 This work was supported by U. S. Public Health Service Grant E-3720, National Institute of Allergy and Infectious Diseases. Presented in part at the Fifth International Congress for Electron Microscopy, Philadelphia, Pa., August 29-September 5, 1962. :! Present address: Department of Pathology, iMedical College of Virginia, Richmond, Virginia. 19
so
FIG. Lilxxal).
K.
1.
13. FRITZ.
G. W.
GANDKH,
.AKl)
17. GO01~A1,li
Rabbit Irucoq~te aitel- incubation it;! I huur \qith Iwtcrial cndutuxin (O..j!Lg oi Above is part of a platelet ; below. part oi the leucoqtr cytoplam and nucleus. Rctween them is a 100x agarcgate of line granules. Apparent defects in Icucocyte plasma membrane, such as seen here: are not necessary related to granular aogrepates. Rounded bodies in and around agpregate are probably cross section5 of cytoplasmic processes. Unstained. X 50.000.
FIG. .3. Portion of leucocyte from a patient febrile with urinary tract infection. So incubation with bacterial endotoxin. Aggregates of granular material apparmtly within cell vacuoles ma) have been phagocytized, may have been formed within the vacuole, or may, because of the plane 01 section, actually be outside the cell. Appearance of granular material here is similar lo that in rabbit and human leucocpte preparations artificially stimulated i11 aitro with bacterial endotoxin (Figs. 1 and 2). Size of individual granules formin p argreaatcs _ in FiKs. l-3 measures from SO to loo.&, although it is obvious that these mcasuremcnts can only be approximate in such a preparation and at such a magnification. Unstained. X 50,000.
RABBIT
LEUCOCYTIC
23
PYROGEN
been made, but confirmatory evidence is accumulating. Some of this evidence has come from the study of chemically purified rabbit leucocytic pyrogen by electron microscopy. This report describes the electron microscopic appearance of purified rabbit leucocytic pyrogen, and compares it morphologically with crude rabbit leucocytic pyrogen. We have examined rabbit pyrogen initially in preference to human pyrogen because it is easier to obtain in quantity and therefore in more pure form. Also, preliminary studies in this laboratory indicate that human and rabbit leucocytic pyrogen are chemically similar (Gander and Goodale, 1962b). MATERIALS
AND
LEUCOCYTE
METHODS
PREPARATIONS
Leucocytes seen in Figs. l-3 were obtained and prepared as previously (Goodale et al.. 1962a). PURIFICATION
OF RABBIT
LEUCOCYTIC
described
PYROGEN
The materials and methods used in partially purifying rabbit leucocytic pyrogen have been described elsewhere in detail (Gander and Goodale, 1962a). Briefly, the crude pyrogen, obtained from the leucocytes of sterile peritoneal exudates, is dialyzed against distilled water, freeze-dried, and applied in phosphate buffer first to a DEAE (diethylaminoethyl) cellulose column, which removes most of the protein, and then to a phosphorylated cellulose column which adsorbs leucocytic pyrogen while allowing virtually all of the remaining protein to be eluted. The pyrogen, in turn, is eluted by a stepwise increase in pH with a resulting eightyfold increase in pyrogenic activity per gram of protein as compared with the crude pyrogen. This pyrogenic protein is inactivated by heating at 60°C for 5 minutes or by trypsin digestion. It is electrophoretically homogeneous at pH’s 5.0 and 8.0. PREPARATION
Direct application
OF
PURIFIED
PYROCEN
FOR
ELECTRON
MICROSCOPY
of pyogen to grid
.d. The first method used was to transfer a very small drop of the pyrogenic protein solution to a Formvar-coated grid by means of a capillary tube, and to allow it to dry. The grid was then exposed to osmium tetroxide vapor for 15 minutes. B. Later, an adaptation of a method described by Porter was used and gave much better results (Porter and Hawn, 1949). Glass slides were cleaned twice in 95% ethanol, dried each time with a lint-free cloth, immersed momentarily in a recently filtered solution of 0.15% Formvar in ethylene dichloride, removed, and allowed to drain and dry in a dust-free area. Using a capillary tube, a thin film of the pyrogenic protein solution was spread over the surface of the Formvar, any excess drained, and the glass slide exposed while wet to osmium tetroxide vapor in a water saturated atmosphere in a covered Petri dish for 15 minutes. The Formvar-coated slide was then removed, the surface rinsed with distilled water, and with a sharp needle the Formvar film was cut into segments approximately 2 mm square. The squares were then floated off the slide onto the surface of distilled water and picked up on acetonecleaned grids raised from below the surface of the water. After drying at room temperature the grids were examined with the electron microscope.
24
K.E.FRITZ,G.
W.GANDER, AND F.GOODALE
C. The pyrogenic protein was also examined after preparation by the negative staining method of Brenner and Horne (1959). A drop of a solution composedof equal parts of 2% aqueous phosphotungstate solution (pH 7.4) and a solution of the pyrogenic protein in phosphate buffer was applied directly to Formvar-coated grids. After drying, the grids were ready for examination, although in someinstances the grids were exposed to osmium tetroxide vapor for 15 minutes prior to examination. D. Protein solution was applied to Formvar-coated grids, air dried, then either exposed to osmium tetroxide vapor for 15 minutes or stained with 0.1% phosphotungstic acid, and shadowed with chromium by an adaptation of Hall’s method to available equipment (Hall, 1949). THIN-SECTION
PREPARATIONS
OF PYROGEN
To prepare samples of pyrogenic protein for thin sectioning, a solution of the protein was first evaporated to dryness in a small glasstube, leaving a minute amount of barely visible, soft, white powdery residue adhering to the walls of the tube. Prepolymerized methacrylate (7 parts butyl methacrylate to 1 part methyl methacrylate) with catalyst ( 1.5% benzoyl peroxide) was introduced into the tube, using great care not to agitate the powder, and the methacrylate hardened at 50°C for 1 week. Following this, the glasstube was broken carefully, pieces of glasswere peeled away from the methacrylate mass, and the methacrylate then examined by phase microscopy. Wherever inclusions of the powdery residue in the methacrylate were found they were cut out and re-embedded in methacrylate in gelatin capsules. Following polymerization, thin sections were cut with glass knives on a Servall Porter-Blum microtome, mounted on Formvar-coated grids, and exposed to osmium tetroxide vapor for 15 minutes. CONTROLS
Protein-free buffer solutions of the type used in the purification of the protein were passedthrough both cellulosecolumns and prepared for the electron microscope as described above in Part B under Direct application of pyrogen to grid. Thin sections of buffer were also prepared as described under Thin-section preparations of pyrogen. All grids were examined with a SiemensElmiskop I electron microscope operated at 60 KV at instrumental magnifications up to 40,000 X using a double condenser lens system with a beam diameter of 5 u. RESULTS DIRECT APPLICATION
OF PYROGEN TO GRID
.4. When the solution containing pyrogen was transferred directly to a Formvarcoated grid, allowed to dry, and then exposed to osmium vapor, examination revealed areas of two types. One contained large, dense,black amorphousprecipitates in which no detail could be discerned. The other showed less dense,finely granular precipitate which bore a striking resemblanceto the granular material seen in pyrogenic white cell preparations. It was difficult, however, to distinguish by this method morphological characteristics of the individual particles which composethe precipitate.
H. In contrast. micrographs of preparations, made by subjecting a thin wet ftlm of Ixotein solution on a Formvar-coated grid to osmium vapor in a water-saturated atmosphere. showed the protein to be made up of discrete. densely osmiophilic dots or particles of about 2.5 A diameter (Fig. 4). They were separated II); a relativel!- con-
stant minimum distance, the distance from dot center to dot center being appro.ximately 50 A. The area between the densely osmiophilic dots was faintly osmiophilic when compared with the background. Excellent dispersion was achieved by this preparative method probably because only the fixed particles directly adjacent to the Formvar film adhered to the lilm through the washing process. Thus the layer ot adherent particles approached a monolayer and sul,eriml,ositioll was minimized. Measurement of the particles was probably more nearly representative of their actual dimensions than would be true if the protein film were allowed to dry before being exposed to osmium vapor. Stresses of dryin: tend to ilatten particles into the supand decreasing their vertical dimensions. portin, 1~ film. thus increasing their horizontal To some extent. at least, it is likely that the iisation of the particles while in solution helps them to withstand dryin, (J with less distortion. (‘. Electron micrographs of the negatively stained protein revealed areas in which the dense black phosl,hotunestate background outlined discrete: unstained spheres (Fig. j) averaging approximately 50 ;i in diameter. So dense, black dots appeared of negatively stained protein in this type of preparation. However. in micrographs
prepared in exactly the same way but then subjected to osmium tetroside vapor: dense, black dots now appeared in the center of the unstained spheres (Fig. 6). Measurements of the dots and of the unstained areas corresponded closely to the measurements of the dots and spacing as shown in the other preparations. Negative staining, however, also brought out the fact that the spheres with their central dots tended to occur in clusters or aggregates of 20-30.
YlG. 5. Pyrogen-containing rxposure to osmium vapor. unstained spheres of about
negatively stained with phosphotunjistic solution JIknse T-shaped hack~round of phosphotun&ic acid 50 i\ diameter.
acid, without outlines indkidual
II. Examination of shadowed preparations revealed some spherical particles. the smallest of which measured about 45 A in diameter. However, the grain size of the deposited chromium metal was so large that the protein particles did not appear in sufficient contrast to warrant conclusions being drawn as to their structure or dimensions. With the availability of more versatile vacuum evaporating equipment, information of considerable import should be obtainable from shadowed preparations.
Micrographs of thin-sectioned preparations of the pyrogen (Fig. i ) again showed densely osmiophilic particles of about 25 A diameter, separated by a relatively constant distance of 50a from particle center to particle center. This appearance is thus the same as that demonstrated when the protein is not thin-sectioned but applied directl!: to the grid surface.
FIN:. 6. Preparation idenlical to that seen in FiC. 5. and thrn exposed 10 osmium u~lor. 111 the center of the .50 .k spheres a densely osmiophilic dot of about 35 A diameter has appeared. .is in Fig. 5 the 50 .% spheres tend lo appear in TWUJX ~ Of d,OUt .
FIG. 7. A thin section Section exposed to fxmium
of methac~);late-cmI,edded vapor. .+T-era;r particle
r&due from p)ru~en-contaillin~ size is about 25 A.
solution.
28
K. E.FRITZ>
G. W. GANDER, CONTROL
AND
F.GOODALE
PREPARATIONS
Micrographs of protein-free buffer solutions that had been passed through both cellulose columns, then thin-sectioned or applied directly to Formvar-coated grids and exposed to osmium tetroxide vapor, showed occasional crystals, but nothing resembling the densely osmiophilic particles of the preparations containing pyrogen. DISCUSSION Both crude and purified leucocytic pyrogen, when stained only with osmium, appear under the electron microscope as particles of two sizes: the smaller and more numerous measureabout 25 A and the larger, 700-800 ii in diameter. In both pyrogens the distance from each small particle center to the center of its neighbor is remarkably constant at about 50 A. The particle size by these measurementswas puzzling becausewhen we calculated the molecular weight for particles of 25 A diameter (assuming a density of 1.3 for dry, unfixed protein) we arrived at a figure of 6000. Because the pyrogenic protein is nondialyzable, such a molecular weight would be quite unreasonable. However, when the pyrogen was negatively stained, its basic structural unit was clearly not a 25 ii particle but a sphere 50 A in diameter in the center of which, when osmium staining was added to the negative staining, a densely osmiophilic core of 25 A diameter appeared. Now we could explain why the smallest unit or particle of the crude osmiumstaIned pyrogen (for which we do not yet have a negative staining technique) appears to be 25 ii in diameter and also why there exists the constant distance of 50 A separating these small particles. In addition, a basic unit of 50 A diameter would have a molecular weight of about 50,000, which is reasonable in view of our knowledge of the chemical properties of the pyrogen. Negative staining of the purified pyrogen also re-emphasizesthat the 50 A spheres tend to form aggregatesof 600-800 A in diameter. In a morphologic study such as this, interpretation of results must be made with extreme caution, perhaps even skepticism. Before assuming that we have indeed visualized moleculesof leucocytic pyrogen we must consider these possiblesourcesof error: I. The granular material seen in leucocyte preparations (which contain crude pyrogen) could be preparative artifact or cell product other than leucocytic pyrogen. However, in a previous publication we have stated our reasons for believing that the particles do, in fact, represent crude leucocytic pyrogen (Goodale et al., 1962a). 2. The particles we have visualized in preparations of purified pyrogen may not be pyrogen but another protein (or proteins) so similar in chemical and physical properties as to be inseparable from the pyrogen by our present purification procedure. This is possible even though the purified protein is electrophoretically homogenous at several different pH values. 3. The particles which we have shown in purified pyrogen preparations might represent a contaminant acquired during purification. The best evidence against this possibility is that buffer solution subjected to the samepurification processdoes not show the osmiophilic particles characteristic of the pyrogenic fractions. Added evidence against their being a contaminant is suggestedby the absenceof the characteristic particles in the nonpyrogenic fractions of the column eluate so far examined.
RABBIT
LEUCOCYTIC
PYROGEN
29
4. Perhaps the particles we are visualizing are a result of preparing the protein for electron microscopy rather than being the size and shape of the protein itself. The most cogent reason for believing that this is not so is that, regardless of the method of preparation (be it thin film in osmium tetroxide vapor, thin section, or negatively stained), the resultant particles are remarkably consistent in appearance and dimensions. 5. The spheres (if we assume 3 dimensions) of 50 j, diameter that we have demonstrated may not be molecules but instead either agglomerates of smaller molecules or fragments of larger molecules. These possibilities cannot be ruled out at present. 6. Morphological similarities between particles seen in pyrogenic cell preparations and those seen in preparations of purified pyrogen obviously do not mean that the two are identical. At best the evidence linking them is circumstantial, and rests on the fact that they appear in pyrogenic preparations and not in nonpyrogenic ones. After considering these possible misinterpretations of our electron micrographs we believe that we have visualized a protein (or proteins) which is either leucocytic pyrogen or protein thus far indistinguishable from it. The particle size is such that it might reasonably represent the intact pyrogen protein molecule. Some evidence suggests that the particulate substance in pyrogenic cell preparations may be the same substance visualized in preparations of purified pyrogen. Assuming that we have visualized the pyrogen molecule. two points of interest arise. First, the molecular weight of 50,000 calculated from the size of the molecules is helpful in our study of a substance which is available in such small quantities. It also seems reasonable in the light of what we know of the properties of the pyrogen. The second point of interest is the chemical and biological significance, if any, of the densely osmiophilic core. The possibility that the site of pyrogenicity may be associated with it is intriguing. Also it may represent a structural concentration of certain amino acids. Of the seven amino acids thus far tentatively identified in the pyrogen. three (tryptophan, arginine, and histidine) are known to react strongly with osmium tetroxide, at least in the test tube (Bahr, 1954). The most feasible approach to positively identifying the visualized protein as the pyrogen is immunologic. If we can successfully produce specific antibodies to the pyrogen, as we are now attempting to do. we will have available a tool of greatest potential for our study. The possible labeling of such antibodies with electron dense substances such as ferritin or uranium should help us localize and identify the pyrogen by electron microscopy in conjunction with immunochemical studies. SUMMARY We have studied by electron microscopy a pyrogenic protein derived from rabbit leucocytes. Whether the pyrogen is in crude or purified form. the basic unit appears the same: a sphere jo A in diameter which has a central, densely osmiophilic core of about 25 A diameter. The reasons for believing that these particles may represent the substance we know chemically as leucocytic pyrogen are: (1) they are consistently present in preparations known by biological or chemical assay to contain leucocytic pyrogen; they are consistently absent in nonpyrogenic controls, (2) The calculated molecular weight of the particulate material (50,000) is consistent with our knowledge of the chemical properties of the pyrogen. (3) We cannot find any preparative artifact or other substance that this particulate material might reasonably repesent. Quite obviously none of our 3 reasons constitutes proof that what we are seeing is really the pyrogen. Although it seems probable to us that we are visualizing the pyrogen, we must wait until we develop an antibody to the pyrogen before we can present more convincing evidence,
30
K.
E.
FRITZ,
G. W.
GANDER,
AND
F.
GOODALE
REFERENCES BAHR, G. F. (1954). Osmium tetroxide and ruthenium tetroxide and their reactions with biologically active substances. Exptl. Cell Res. 7, 459-479. BEESON, P. B. (1948). Temperature-elevating effect of a substance obtained from polymorphonuclear leucocytes. 1. Clin. Invest. 27, 524. RENNET, I. L., JR., and BEESON, P. B. (1950). The properties and biologic effects of bacterial pyrogens. Med. 29, 365-400. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. Biochem. Biophys. Acta 34, 103-110. CRANSTON, W. I., GOODALE, F., SNELL, E. J., and WENDT, J. F. (1956). The role of leucocytes in the initial action of bacterial pyrogens in man. Clin. Sci. 16, 2 19-226. GANDER, G. W., and GOODALE, F. (1962a). Chemical properties of ieucocytic pyrogen. I. Partial purification of rabbit leucocytic pyrogen. Exptl. Mol. Puthol. 1, 417-426. GANDER, G. W., and GOODALE, F. (1962b). A method for purification of human leucocytic pyrogen. Proceedings of the Ninth Congress of the International Society of Hematology, Mexico City, September (Abstract). GOODALE, F., FILLMORE, R., and HILLMAN, E. (1962a). Electron microscopic observations of human leucocytes. I. Response in vitro to bacterial endotoxin. Ewptl. Mol. Pathol. 1, 229-250. GOODALE, F., HILLMAN, E., and FILLMORE, R. (1962b). Observations of human leucocytes from patients with naturally occurring fevers. In “Proceedings of the Fifth International Ccngress for Electron Microscopy” (S.S. Breese, ed.), Vol. 2, p. SS-3. Academic Press, New York. HALL, C. E. (1949). Electron microscopy of fibrinogen and fibrin. J. Biol. Chem. 179, 857-864. MENKIN, V. (1943). Chemical basis of injury in inflammation. Arch. Pathol. 36, 269-288. PORTER, K. R., and HAWN, C. Z. (1949). Sequences in the formation of clots from purified bovine fibrinogen and thrombin: A study with the electron microscope. J. Exptl. Med. 90, 225-232. RAFTER, W. S., COLLINS, R. D., and WOOD, W. B., JR. (1960). Studies in the pathogenesis of fever. VII. Preliminary chemical characterization of leucocytic pyrogen. J. Exptl. Med. 111, 831-840. SNELL, E. S., GOODALE, F., WENDT, F., and CRANSTON, W. I. (1956). Properties of human endogenous pyrogen. Clin. Sci. 16, 615-626. SNELL, E. S. (1962). Pyrogenic properties of human pathological fluids. CZin. Sci. 23, 141.150. WAGNER, R. R., BENNETT, I. L., JR., and LEQUIRE, V. S. (1949). The production of fever by influenza1 viruses. I. Factors influencing the febrile response to single injections of virus. Z. ExjtZ. Med. 90, 321-334.