Chemical and Morphological Studies Could Be Made of Polysomes of Lymphoid Tissue Cells During Response to Antigen Injection

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THE NA TURE OF POLYSOMES ISOLATED FROM SPLEEN CELLS OF RATS STIMULATED BY ANTIGEN* BY MARIANO F. LA VIA, t ALBERT E. VATTER, WILLIAM S. HAMMOND,t AND PATRICIA V. NORTHUP

DEPARTMENT OF PATHOLOGY, UNIVERSITY OF COLORADO MEDICAL CENTER, DENVER Communicated by Paul R. Cannon, November 7, 1966 Kern and co-workers' were the first to examine the molecular mechanism of immunoglobulin synthesis and concluded that this process is similar to the synthesis of other proteins. Recently, lymphoid tissues have been studied by more refined electron microscopic2' I and biochemical techniques4- in an effort to examine details of the mechanism of synthesis of antibody. The data presented in many of these studies are difficult to reconcile with present models of polypeptide synthesis on polysomes. The current hypothesis is that the size of a fully functional polysome should be compatible with the size of the product of synthesis.7 Thus for L and H chains one would expect polysomes in the range of 7 and 15 ribosomes, respectively. The examination of polysomal units of immunoglobulin synthesis isolated from lymph node and spleen homogenates has been complicated by the presence of high- activity ribonucleases (RNases). These tissues seem to lack the RNase inhibitors present in liver cells.8 The contradictory results of experiments in this field might be due to endogenous RNases. On the other hand, synthesis of an antibody molecule might proceed differently from the model proposed for other polypeptides. The work reported here is the result of efforts to develop a system in which biochemical and morphological studies could be made of polysomes of lymphoid tissue cells during response to antigen injection. The first objective was to devise a means to reduce the degradative action of endonucleases on the preparations being studied. The initial effort to develop such a system was partially successful.9 The experiments presented here indicate success in devising a method to block RNase action completely in homogenates of spleen and lymph nodes. Interpretation of the results of these experiments suggests that some discrepancies may exist between accepted models of protein synthesis and the types of polysomes involved in the assembly of L and H chains of antibody globulins. Materials and Methods.-Animals and immunization: Sprague-Dawley male albino rats weigh- ing between 150 and 300 gm were used in all experiments. They were immunized in two ways: primary immunization consisted of an intravenous injection of 1.0 ml of Salmonella typhi formalin- killed vaccine;10 hyperimmunized animals were given three intravenous injections of 1.0 ml of the vaccine on 3 consecutive days and 30 days later received a fourth intravenous injection to serve as a booster. No differences were noted in the results of experiments using rats given either immunizing treatment, and therefore no distinction will be made between hyperimmunized and nonhyperimmunized animals. Isotopes: Reconstituted, C14-labeled Chlorella hydrolysate prepared by Schwarz BioResearch was used in all experiments. Cell preparation, incubation, and centrifugation: Rats were killed by exsanguination under ether anesthesia; the spleen was rapidly removed, placed in ice-cold buffered special saline (BSS, 0.03 M tris, 0.14 M NaCl, 0.005 M KCl, 0.0015 M MgC92), and finely minced. Splenic fragments were then forced gently through an 80-mesh stainless-steel screen by means of a syringe plunger, with frequent BSS washings. Collected cells were washed twice by centrifugation at 250 X g in cold BSS with ethylenediaminetetraacetate (EDTA) and then in cold BSS without EDTA. The cell suspension from one spleen was then incubated in a Dounce homogenizer vessel in 2.0 ml of Eagle's 79 Downloaded by guest on September 24, 2021 80 PATHOLOGY: LA VIA ET AL. PROC. N. A. S.

minimal medium with 1/1o the normal concentration of amino acids. Incubation was continued for 15 min at 370C and 20 ise of C14-labeled Chlorellk hydrolysate were added. Incubation was continued for 2-3 min and 10 ml of ice-cold BSS were added to stop the reaction. Cells were washed twice with cold BSS by centrifugation at 250 X g. One milligram of splenic ribonucleic acid (see below under ribonuclease assay and inhibition), 0.04 ml of bentonite (8000 X g particles), and 0.1 ml of a 1%1 solution of BRIJ 58 (polyoxyethylene cetyl ether, Atlas Chemical Co.) were added in 1.0 ml of medium A (0.01 M Tris, 0.01 M KCl, 0.015 M A1gCl2, pH 7.4) with 0.25 M sucrose, and brief homogenization was carried out (5-6 strokes with a tight-fitting pestle). The contents of the homogenizer were transferred to centrifuge tubes and centrifuged at 17,000 X g for 15 min to remove cell debris, nuclei, lysosomes, and mitochondria. The supernatant was layered on a 0.3-1.0 M sucrose density gradient and centrifuged at 113,000 X g (maximum) for 210 min in an SW25.3 rotor in a model L-2 Spinco centrifuge at 0C. Gradient examination: At the end of centrifugation the bottom of each tube was punctured, and optical density at 260 mjA was determined by allowing the preparation to flow through anl LKB quartz cell (4-mm light path) continually monitored by means of a Beckman DU mono- chromator in combination with a Gilford model 220 photocell-optical density converter attached to a Honeywell recorder. Between 25 and 35 fractions were collected and 1-drop samples from several fractions were taken for electron microscopic examination and frozen. To the remaining material in each tube were added 0.2 mg of BGG and trichloracetic acid to a final concentration of 5%. After at least 30 min at 40C, precipitates were collected on HA Millipore filters and washed with excess 5% TCA. Filters were glued onto aluminum planchets and counted for 10 min each in a Tracerlab, low-background gas-flow counter (background maximum 2.0 epm). Titration of sera and supernatants: Rat sera were obtained at the time of sacrifice in several experiments, and agglutinin titers were determined by a method described previously'1 before and after treatment with 2 mercaptoethanol at a final concentration of 0.05 lMI. The supernatant remaining after cell incubation in experiments carried out on S. typhi-immunized rats was assayed for newly formed antibody. After separation on a CM-Sephadex C50 column (1.0 X 25 cm, equilibrated with 0.01 M P04 buffer with 1 mg/ml KCN pH 6.8) to remove hemoglobin, the y- globulin was precipitated with 50% saturated ammonium sulfate and redissolved in saline. A 1.0-ml aliquot of vaccine was added and the sample was incubated at room temperature for 1 hr and at 370C for 10-20 hr. The bacteria were then plated on aluminum planchets which were counted as described above. Radioactivity bound to bacteria indicates newly synthesized antibody. Material from control rats treated similarly did not yield antigen-binding radioactivity. Ribonuclease assay and inhibition: Since ribonuclease, liberated from damaged or broken lysosomes, might be responsible for polysome breakdown, precautions were taken to protect polysomes and assay of the enzyme was done randomly. Assay for ribonuclease activity was done by using rat liver C ribosomes prepared as described by Wettstein et al.'2 To 0.5-ml aliquots of C ribosomes one of the following was added: 0.1 ml of supernatant to be assayed or 0.1 ml BSS containing 10-3 ,ug/ml pancreatic crystalline ribonuclease (Worthington). The aliquots were incubated for 10 min at 0C and were centrifuged in a sucrose density gradient under the conditions outlined above. A sample of untreated C ribosomes was also centrifuged. Optical density at 260 m1A was then measured (see above). Ribonucleic acid added to cells before homogenization was prepared from normal rat spleen by blending this organ in phenol saturated with Tris buffer, pH 5, at room temperature. The homog- enate was heated at 650C until it reached a temperature of 630C. It was then frozen in dry ice- acetone bath, thawed rapidly, and centrifuged at 1100 X g for 10 min. The aqueous phase was removed and the interphase was re-extracted at room temperature. The two aqueous extracts were combined and RNA was precipitated by addition of absolute ethanol 2: 1 (v/v). The RNA was stored in this state at -20'C. Preparations of RNA of this type gave a 260/280 OD ratio of 1.9. Density gradient centrifugation was performed on a sample of this RNA. The typical peaks usually formed in undegraded RNA were seen by optical-density scan of the gradient: very high 288, intermediate 18S, and very low 4S. Electron microscopic studies: Samples for electron microscopy were prepared by placing a drop of a 1 to 10 dilution of the preparation to be examined on a collodion-coated grid. After a few seconds the grid was washed with 0.25 M sucrose in BSS and then with water, blotted, and shadowed with chromium at an angle of 4 to 1. The preparations were examined with a Phillips 200 electron microscope. Downloaded by guest on September 24, 2021 VOL. 57, 1967 PATHOLOGY: LA VIA ET AL. 81 Results.-The results of a typical experiment are illustrated in Figure 1. For reasons which are still unexplained, the RNA added to each sample to protect it from ribonuclease action masked the optical density profile obtained in scanning the gradients so that no distinct peaks of optical density could be observed. Therefore only the results of radioactivity determinations are shown. Radioactivity incorporated by spleen cells from nonimmunized rats into nascent polypeptide chains is indicated by the dotted line. The labeled polysomes from immunized animals are 80 x-x 3 DAYS AFTER 190 S IMMUNIZATION x CONTROL 75 ono j UNIMMUNIZED IT x 25

20 -

a.02 I5 Il 10

5 10 15 20 25 30 FRACTION NUMBER FIG. 1.-Radioactive amino acids were incorporated into nascent protein in isolated spleen cells of rats which had been immunized with S. typhi 3 days prior to sacrifice (x) and of control rats (o). The experimental conditions are described in the text. While the two patterns are generally similar, there is a marked difference in the level of incorporation in control vs. immunized rat spleen cells. Three groups of polysomes appear to be active in immune animal cells, 190S, 266S, and 320S. represented by the solid line. In normal rats two peaks are seen; these are at 80S and 118S and represent monomers and dimers. This suggests that small amounts of nascent protein are associated with these ribosomes. In addition to these peaks there is a broad, flat hump with a maximum approximately at 230S, indicating some synthesis of larger polypeptides. A high radioactivity content is present in heavy material in the first two gradient fractions. This is probably due to heavier mem- brane-bound polysomes. The immunized rats by comparison show prominent peaks in the 190S, 266S, and 320S areas. A shoulder of material heavier than 320S showing radioactive amino acid incorporation is also present. The experiment Downloaded by guest on September 24, 2021 82 PATHOLOGY: LA VIA ET AL. PROC. N. A. S.

25/

360 S 20 x

L 15 202OS

I 0 I/N- 80X..5X 5 X Xx#x JX

p 8 ~ ~~A ~a 5 10 IS 20 25 30 35 FRACTION NUMBER FIG. 2.-Synthesis of nascent protein on polysome of spleen cells of a rat immunized 1 month prior to sacrifice and re-injected 4 days before sacrifice. Experimental procedure is described in detail in the text. There are two small peaks of radioactivity at the 80S and 120S regions repre- senting protein chains on monomers and dimers. One higher peak at 360S and a series of peaks culminating in the 200S area are compatible with the synthesis of heavy (H) and light (L) chain subunits. illustrated in Figure 2 was carried out as the previous one (see Fig. 1). However, a rat injected four days before sacrifice was used. While more fractions were collected, the pattern observed with animals sacrificed three days after antigenic stimulation (see Fig. 1) is apparent. Small peaks of monomers (80S) and of dimers (120S) are present and, in addition, there are two classes of polymers with peaks in the 200S and 360S areas. The latter peak is very narrow and therefore sup- posedly made up of polysomes of very uniform size. On the contrary, the 200S peak is fragmented in many smaller and lower peaks and represents the top of a region of gradient extending from trimers to hexamers. The supernatant fraction in each case contains very highly labeled material. This could be synthesized protein or free amino acids from the cell's amino acid pool. Under the conditions of these experiments, immungolobulin is the major product of synthesis of spleen cells. Because of the considerable extra- medullary erythropoietic activity of the splenic red pulp, hemoglobin is also synthesized. The following results were obtained by fractionating the supernatant on a column of C1\1-Sephadex C5O-counts: in supernatant, 1512; in effluent, 430; in y-globulin, 122; and in antibody, 20 (see Titration of sera and supernatants in Materials and Methods). Under these conditions free amino acids and hemoglobin are retained in the column. The effluent containing the immunoglobulin was treated with S. typhi as outlined above. It is apparent that the y-globulin and immunoglobulin fractions contains a large portion of the label which has been incor- Downloaded by guest on September 24, 2021 VOL. 57, 1967 PATHOLOGY: LA VIA ET AL. 83

porated into newly synthesized protein. Thus it can be concluded that in the system studied, the synthesis of specific immunoglobulin is proceeding actively. Many cells, however, are engaged in the synthesis of other proteins and polysomes synthesizing other polypeptides are definitely present within immunoglobulin- synthesizing cells. Because of the problems inherent in the presence of large amounts of ribonuclease in spleen preparations, random ribonuclease assays were done (see Materials and Methods). The results of these assays always revealed no depolymerization of rat liver polysomes after treatment with spleen supernatant, while some depolymerization was always present after treatment with 10-s Ag crystalline pancreatic RNase. Thus it can be concluded that the addition of spleen RNA during the disruption of cells effectively blocks ribonuclease action. While the reasons for this phenomenon are not apparent, it is possible that the inhibition observed may be due to an excess of substrate in proportion to the amount of enzyme present. The polysomes illustrated in Figure 3 were obtained from fractions in the 190S and 320S areas, respectively. It appears that the 190S polysomes are primarily trimers and tetramers and those in the 320S areas are primarily octamers to decamers. This agrees well with sedimentation coefficients for these two fractions. In this figure, strands of RNase-sensitive material (compatible with the appearance of mRNA) are visible in relation to some of the polysomes. In addition the polysomes show some ir- regularity with occasional spaces where the RNA strand is exposed. It was expected that the strand would be occupied by a continual series of closely arranged ribosomes. Possible reasons for this latter phenomenon will be discussed below. Titration of sera from animals used in these experiments revealed that titers observed after primary immunization were 80 per cent 2 ME-sensitive and that those after secondary stimulation were completely 2 ME-resistant. Discussion.-The results reported above indicate that in normal rat spleen cells there are polysomes that are making protein in small amounts. The study of the subcellular events which accompany protein synthesis has shown that amino acids are assembled in polypeptide chains on ribosomes. The ribosomes are arranged on a molecule of messenger RNA (mRNA) which carries the informa- tion that dictates the sequence of amino acids in the protein. Present evidence indicates that sequences of three nucleotides code for an amino acid.'3 The maximum number of ribosomes which could be located on a strand of mRNA at one time would be limited by the length of the RNA molecule.7" 4 It has been shown that in the case of hemoglobin synthesis the maximum number of ribosomes found on the mRNA is five.7 This unit of protein synthesis comprised of mRNA and ribosomes has been referred to by the terms of polyribosome, polysome, and ergo- some.7, 12 When proteins are being synthesized at maximum rates, it is expected that the mRNA molecule would be completely saturated with ribosomes. Lam- from and Knopf" have shown that mRNA molecules with single ribosomes will synthesize protein. From this observation it appears that whether or not the mRNA molecule is saturated with ribosomes, there is no difference in the product other than perhaps the rate of synthesis. In immunized rat spleen cells, protein is produced in greater amounts and the polysomes active in polypeptide synthesis appear to be segregated primarily in two classes with sedimentation coefficients of about 190-200S and 320-360S, respectively. Downloaded by guest on September 24, 2021 84 PATHOLOGY: LA VIA ET AL. PRoc. N. A. S.

FIG. 3.-Electron micrographs of polysomes isolated by sucrose density gradient centrifugation (see text). (A) A preparation of the 190S polysome formation, and (B) the 310S fraction. It is evident that trimers and pentamers are preponderant in the 190S fraction, while octomers to decamers are abundant in the 320S polysomes. The irregular spacing described in the text is evident in some of the polysomes. Magnification X43,000. Downloaded by guest on September 24, 2021 VOL. 57, 1967 PATHOLOGY: LA VIA ET AL. 85

The synthesis of hemoglobin has been reported to occur as separate a and 3 chains, synthesized on separate polysomes and assembled in the final hemoglobin molecule after detachment from the polysome.'6 Recently a similar mechanism has been described for mouse myeloma globulin L and H chains.'7 In order to synthesize protein molecules of the molecular weights of L and H chains, one would expect the polysomes to be made up of approximately seven and 15 ribosomes, respectively. Using the values found by Pfuderer et al.,'8 the sedimentation coefficient for seven- member polysomes is 257S 4 6S. While figures for polysomes larger than 12 n were not obtained, the S value given for this polysome is 353. The polysomal peak observed in these experiments range from 190-200S to 320-360S, respectively. This would indicate the larger polysomes to have 10-12 ribosomes and the smaller ones to consist of four ribosomes. It is apparent that the products of neither of these polyribosomes are large enough to account for the completely formed L and H chains, although the size of the larger polysomes may be sufficiently close to that expected for a polypeptide of 55,000 molecular weight. Recently published experiments have demonstrated the presence of L chain subunits of approximately 10,000 molecular weight in the urine of multiple myeloma patients.'9 On the other hand, structural studies of Bence-Jones proteins in humans have shown that each of these proteins consist of 212 amino acids arranged so that residues 1 to 106 are unique, while residues 107 to 212 are always arranged in similar order with the exception of one amino acid in position 189 responsible for Inv characters.20-22 These observations in human material have been confirmed and extended in mice with multiple myeloma where it has been shown that differences similar to those observed in L chains also exist in H chains. An under- standing of the assemblage of these complex molecules may aid in the interpretation of genetic mechanisms for the control of immunoglobulin synthesis. One possible interpretation is that the invariable piece of a large number of L chains may be coded for by one gene only, and the variable piece by a large number of genes each responsible for one sequence.23 While this concept awaits further confirmation, the results of the studies reported here appear compatible with it. In the system described, the synthesis of L and H chains is carried out in addition to that of other proteins. Nascent proteins are associated with polysome classes around 200S, 260S, and 320-360S. The S values observed are not compatible with polysomes synthesizing proteins of the size expected but seem to favor the possibility that L and H chains are synthesized as subunits on different polysomes. The morphologic appearance of polysomes in this study agrees with that observed by others. However, while the majority of polysomes appear uniform when viewed with the electron microscope, some irregular spacing of the ribosomes is occasionally present. This could be explained by one of several factors: degrada- tion of some ribosomes by RNase, artifactual assembly of polysomes because of deficiencies in the incubation system, a defect in the chain initiation mechanism leading to irregular entry of ribosomes on the mRNA chain, or a decreased avail- ability of ribosomes during very active synthesis. Unpublished studies of the RNase sensitivity of these polysomes have shown that the first possibility is ex- tremely unlikely unless one invokes special conditions. In fact while the mRNA is easily degraded by treatment with RNase at 0-40C for ten minutes, this treatment has to be prolonged for 30 minutes at 37°C before ribosomal morphology is lost. Downloaded by guest on September 24, 2021 86 PATHOLOGY: LA VIA ET AL. PROC. N. A. S.

More experimentation will be necessary, however, before a conclusion can be reached. Summary. Spleen cells of rats immunized three or four days prior to sacrifice contain polysomes compatible with the synthesis of pieces of H and L chains. Gamma globulin and specific antibody are the primary proteins synthesized in this system. The synthesis of other proteins for cell renewal cannot be excluded.

* This work was supported by Army contract DA18-035-AMC-390, by grant T-290A from the American Cancer Society, and by institutional grant IN-5G from the ACS. t Recipient of a Career Development Award K3-A1-18,421. I Trainee, USPHS training grant 5TlCA5164-02 of the National Cancer Institute. 1 Kern, M., E. Helmreich, and H. N. Eisen, these PROCEEDINGS, 45, 862 (1959). 2 De Petris, S., and G. Karlsbad, J. Cell Biol., 26, 759 (1965). 3Harris, T. N., K. Hummeler, and S. Harris, J. Exptl. Med., 123, 161 (1966). Voss, E. W., D. C. Bauer, and W. R. Finnerty, Life Sci., 4, 731 (1965). Tawde, S., M. D. Scharff, and J. W. Uhr, J. Immunol., 96, 1 (1966). 6 Norton, W. L., D. Lewis, and M. Ziff, these PROCEEDINGS, 54, 851 (1965). 7Rich, A., J. R. Warner, and H. M. Goodman, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 269. 8 Blobel, G., and V'. R. Potter, these PROCEEDINGS, 55, 1283 (1966). 9 La Via, M. F., A. E. Vatter, and P. V. Northup, Exptl. Mol. Pathol. (Suppl.), 3, 124 (1966). 10 La Via, M. F., J. Immunol., 92, 252 (1964). 1 Cannon, P. R., W. E. Chase, and R. W. Wissler, J. Ir;tmunol., 47, 133 (1943). 12 Wettstein, F. 0., T. Staehelin, and H. Noll, Nature, 197, 430 (1963). 13 Khorana, H. G., Federation Proc., 24, 1473 (1965). 14 Gilbert, W., in Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 287. 15 Lamfrom, H., and P. M. Knopf, J. Mol. Biol., 11, 589 (1965). 16 Colombo, B., and C. Baglioni, J. Mol. Biol., 16, 51 (1966). 17 Shapiro, A. L., 1M. D. Scharff, J. V. Maizel, Jr., and J. W. Uhr, these PROCEEDINGS, 56, 216 (1966). 18 Pfuderer, P., P. Cammarano, 1). It. Holladay, and 1). G. Novelli, Biochim. Biophys. Acta, 109, 595 (1965). 19 Cioli, D., and C. Baglioni, J. Mol. Biol., 15, 385 (1966). 20 Hilschmann, N., and L. C. Craig, these PROCEEDINGS, 53, 1403 (1965). 21 Titani, K., E. Whitley, Jr., L. Avogardo, and F. W. Putnam, Science, 149, 1090 (1965). 22Baglioni, C., L. A. Zonta, and D. Cioli, Science, 152, 1517 (1966). 23 Hood, L. E., W. R. Gray, and W. J. Dreyer, these PROCEEDINGS, 55, 826 (1966). Downloaded by guest on September 24, 2021