Studies with human leukocyte lysosomes. Evidence for antilysosome antibodies in erythematosus and for the presence of lysosomal antigen in inflammatory diseases.

D A Bell, … , J H Vaughan, J P Leddy

J Clin Invest. 1975;55(2):256-268. https://doi.org/10.1172/JCI107929.

Research Article

Human lysosomes were isolated from normal peripheral blood leukoyctes and characterized by electron microscopy, enzyme analysis, and assays for DNA and RNA. Stored sera from 37 unselected patients with systemic lupus erythematosus (SLE), including active and inactive, treated and untreated cases, were tested in complement fixation (CF) reactions with these lysosome preparations. 23 SLE sera exhibited positive CR reactions, as did sera from two patients with "lupoid" hepatitis. The seven SLE sera with strongest CF reactivity also demonstrated gel precipitin reactions with lysosomes. Neither CF nor precipitin reactions with lysosomes were observed with normal sera or with sera of patients with drug-induced lupus syndrome, rheumatoid (RA), polymyositis, or autoimmune hemolytic anemia. By several criteria the antilysosome CF and precipitin reactions of SLE sera cound not be attributed to antibody to DNA, RNA, or other intracellular organelles. The lysosomal component reactive with SLE sera in CF assays was sedimentable at high speed and is presumably membrane associated. The CF activity of two representative SLE sera was associated with IgG globulins by Sephadex filtration. A search for lysosomal antigen in SLE and related disorders was also made. By employing rabbit antiserum to human lysosomes in immunodiffusion, a soluble lysosomal component, apparently distinct from the sedimentable (membrane-associated) antigen described above, was identified in serum, , or pleural fluid from patients with […]

Find the latest version: https://jci.me/107929/pdf Studies with Human Leukocyte Lysosomes

EVIDENCE FOR ANTILYSOSOME ANTIBODIES IN LUPUS ERYTHEMATOSUS AND FOR THE PRESENCE OF LYSOSOMAL ANTIGEN IN INFLAMMATORY DISEASES

DAVID A. BELL, PATRICIA A. THIEM, Jom H. VAUGHAN, and JOHN P. LEDDY From the Division of Immunology, Departments of Medicine and Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, and the Division of , Scripps Clinic and Research Foundation, La Jolla, California 92037

A B S T R A C T Human lysosomes were isolated from A search for lysosomal antigen in SLE and related normal peripheral blood leukocytes and characterized by disorders was also made. By employing rabbit antiserum electron microscopy, enzyme analysis, and assays for to human lysosomes in immunodiffusion, a soluble ly- DNA and RNA. Stored sera from 37 unselected patients sosomal component, apparently distinct from the sedi- with systemic lupus erythematosus (SLE), including mentable (membrane-associated) antigen described active and inactive, treated and untreated cases, were above, was identified in serum, synovial fluid, or pleural tested in complement fixation (CF) reactions with these fluid from patients with SLE, RA, ankylosing spondyli- lysosome preparations. 23 SLE sera exhibited positive tis, and leukemoid reaction. An antigenically identical CF reactions, as did sera from two patients with "lu- soluble component reactive with the rabbit antiserum poid" hepatitis. The seven SLE sera with strongest CF could be released in vitro from intact lysosomes by re- reactivity also demonstrated gel precipitin reactions with peated freeze-thaw cycles. Thus, the naturally occurring lysosomes. Neither CF nor precipitin reactions with soluble antigen may be liberated from leukocytes in vivo lysosomes were observed with normal sera or with sera as a normal aspect of , and clearly is not of patients with drug-induced lupus syndrome, rheuma- unique to SLE. In those SLE patients possessing anti- toid arthritis (RA), polymyositis, or autoimmune hemo- lysosome antibodies, however, release of lysosomes at lytic anemia. By several criteria the antilysosome CF sites of inflammation could lead to local formation of and precipitin reactions of SLE sera could not be at- immune complexes with potential for perpetuating tissue tributed to antibody to DNA, RNA, or other intracel- injury. lular organelles. The lysosomal component reactive with SLE sera in CF assays was sedimentable at high speed INTRODUCTION and is presumably membrane associated. The CF ac- tivity of two representative SLE sera was associated The sera of patients with systemic lupus erythematosus with IgG globulins by Sephadex filtration. (SLE)' characteristically contain antibodies reactive with a variety of plasma, cellular, and subcellular con- This work was presented in part at the 35th Annual stituents. Among these, to nuclear anti- Meeting of The Arthritis and Association (1). gens have been the most extensively studied, and DNA: Dr. Bell was a Postdoctoral Fellow of the Medical Re- anti-DNA complexes have been implicated in the de- search Council of Canada and his present address is Uni- versity Hospital, University of Western Ontario, London, velopment of the renal and vascular lesions of this Ontario, Canada. Dr. Leddy was a Senior Investigator of the Arthritis Foundation. A portion of this work was in ',Abbreviations used in this paper: ANA, antinuclear partial fulfillment of the requirements for a Master's degree antibody; CF, complement fixation; EA, sensitized sheep in Microbiology by Miss Thiem. red cells; E: S, enzyme: substrate; RA, rheumatoid ar- Received for publication 2 November 1973 and in revised thritis; RF, rheumatoid factor; SLE, systemic lupus ery- form 22 August 1974. thematosus; SN, supernate; VBS, Veronal-buffered saline. 256 The Journal of Clinical Investigation Volume 55 February 1975a256-268 disease (2-6). Antibodies to several types of cytoplasmic possessed typical morphologic features of lysosomes Recog- antigens have also been described (7-15) but their nizable cells were not observed. Occasional fragments of cell membrane or of other subcellular organelles were pathogenetic importance in SLE is less clear. found in some fields. Previous studies on the occurrence of antibodies to Acid phosphatase, myeloperoxidase, and neutral protease lysosomes in SLE were based upon complement fixa- activities were readily detectable in fresh preparations of tion (CF) reactions with relatively crude cytoplasmic lysosomes but were greatly increased by disruption of fractions derived to a large extent from nonhuman the granules with 0.1% Triton X-100. Acid phosphatase tis- was measured as described by Janoff, Weissman, Zwei- sues (10, 16). The present investigation was designed fach, and Thomas (19) with p-nitrophenyl phosphate as to reexamine the question of antibodies to lysosomes in substrate. Myeloperoxidase was assayed with O-dianisidine SLE, with particular respect to: (a) immune reactivity as substrate, according to Klebanoff (20). Neutral protease of SLE sera with extensively characterized preparations activity was assayed by digestion of denatured hemoglobin (21, 22) but in a pH 7.3 phosphate-buffered saline system of human lysosomes by both CF and immunodiffusion instead of acid buffer. p-Glucuronidase was also measured techniques; and (b) the possible presence of lysosomal in such lysosome preparations when the method of isolation antigen(s) in the sera or pathologic fluids of SLE pa- was being developed (17). Succinic dehydrogenase (23) tients. For comparison, similar studies were also under- and reduced NAD dehydrogenase (cytochrome c reduc- tase) (24) activities were not detected in the current taken in patients with other rheumatic or immunologic lysosome preparations. disorders. (Na+, K+)-ATPase activity was measured in lysosome membranes as a test for contamination by fragments of METHODS leukocyte plasma membrane (25). This assay, kindly per- Preparation and characterization of antigens formed by Dr. F. H. Kirkpatrick, Department of Radia- tion Biology and Biophysics (Rochester), is based on Lysosome granules were prepared from the peripheral cleavage of [,y-82P]ATP in a sensitive modification' of blood leukocytes of several normal group 0 donors by a the method of Berenblum and Chain (26). ATPase ac- slight modification of the method of Chodirker, Bock, and tivity was measured in the presence of 1 mM Tris ATP Vaughan (17). 100 ml of freshly drawn venous blood was (pH 7.2), 1.5 mM MgCl2, 5 mM HEPES (N-2-hydroxy- immediately diluted in 1,900 ml of cold isotonic saline ethylpiperazine-N'-2-ethane sulfonic acid) buffer (pH 7.2), containing 0.1% bovine serum albumin and subjected to and either 65 mM NaCl or 50 mM NaCl plus 15 mM cold centrifugation in 250-ml glass centrifuge bottles (800 KC1. Inhibition was tested with 10-' M ouabain. For this g, 30 min) to recover the cells. After three cycles of hypo- procedure lysosomes from two preparations (672 and 400 tonic saline lysis of red cells, the remaining leukocytes ,ug protein, respectively) were subjected to 10 cycles of were recovered by cold centrifugation (110 g, 5 min) and freezing and thawing followed by sedimentation of the then washed successively in cold isotonic saline and 0.34 M membranes at 31,000 g for 30 min and three washes in 20 sucrose. The washed leukocytes were lysed in 0.2 M sucrose vol 0.34 M sucrose. Background ATPase was readily containing 3.6 mg (500 U) per ml of heparin (17). Over- demonstrated in both preparations of lysosome membranes night cold incubation (40C) of leukocyte lysates was in the presence of Mg, (240±8 and 180±8 nmol/mg pro- followed by low-speed centrifugation (800 g, 15 min) to tein/h, respectively). Significant (Na+, K+)-dependent remove cell debris and this was followed by centrifugation ATPase activity was not detected (< 17±t8 and < 16±12 (25,000 g, 10 min) at 4VC to sediment the lysosome gran- nmol/mg protein/h, respectively), either as stimulation of ules. The granules were resuspended in 0.34 M sucrose Mg++-ATPase by Na++K+, or as inhibition by ouabain of (containing 250 U of heparin per ml), washed in heparin- Mg++-dependent or (Na+, K+) -stimulated activity. free 0.34 M sucrose, then cleared of lysosomal clumps Lysosomes were also obtained by an alternative protocol (centrifugation at 600 g, 10 min) before use. All centri- obtained from Dr. Robert I. Lehrer, University of Cali- fugations were carried out in refrigerated centrifuges (PR fornia. Briefly, heparinized blood was subjected to (a) 2, International Equipment Co., Needham Heights, Mass.; low-speed centrifugation to remove platelets and plasma; RC 2B, Ivan Sorvall, Inc., Newtown, Conn.; L-240, Beck- (b) centrifugation at 400 g in a Ficoll-34%o Hypaque man Instruments, Inc., Spinco Div., Palo Alto, Calif.). gradient, yielding a mononuclear layer (discarded) and a The final yield of lysosome granules in, such prepara- granulocyte-red cell pellet; (c) sedimentation of the latter tions was 3-4 ml of a suspension containing approximately fraction in 3% dextran followed by hypotonic lysis to re- 1 mg protein/ml by Folin analysis using an egg albumin move red cells; (d) resuspension of granulocytes in 0.34 standard (18). The preparations were promptly examined M sucrose and homogenization in a glass-Teflon homoge- by phase-contrast microscopy (1,000 X) and typically nizer; (e) washes in 0.34 M sucrose to remove debris and showed a myriad of granules dancing freely in Brownian final recovery of lysosomes by centrifugation in cellulose movement. Granule preparations were discarded if they nitrate tubes in a Spinco model L ultracentrifuge (SW-50 contained recognizable red cells, leukocytes or platelets, or rotor) for 20 min at 27,000 rpm. The yield of lysosomes in more than occasional cell debris or granule clumps. As our hands was comparable to that in our standard proce- a further test of lysosomal purity, electron micrographs dure. The second method was selected because it avoids of two representative preparations were kindly prepared exposure of subcellular structures to high concentrations by Dr. Goetz W. Richter, Department of Pathology, Uni- of heparin, which has been shown to release DNA and versity of Rochester. Lysosomal granules were sedimented RNA from rat liver nuclei (27). (25,000 g, 10 min), fixed with 2% gluteraldehyde (3 h) and 1% 0304 (1 h), and stained with uranyl acetate (20 2Kirkpatrick, F. H., G. M. Woods, and P. L. LaCelle. min) and lead citrate (5 min). These electron micro- 1974. Multiple ATPase activities of spectrin: stimulation by graphs (Fig. 1) revealed that virtually all of the particles calcium and magnesium. Submitted for publication. Lysosomal Antigen and Antibody in Lupus Erythematosus 257 . t

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FIGURE 1 Electron micrograph of lysosome granules isolated from human peripheral blood leukocytes. X 58,000.

258 D. A. Bell, P. A. Thiem, J. H. Vaughan, and J. P. Leddy Assay of lysosome preparations for DNA was performed 1/160 Dilution 1/2,560 Dilution by the diphenylamine method with caif thymus DNA (Worthington Biochemical Corp., Freehold, N. J.) as standard (28). RNA was kindly assayed by Dr. Arnold Meisler, Departments of Medicine and Microbiology (Ro- so chester), using the method of Scott, Fraccastoro, and Taft Normal o (29). Soluble nucleoprotein and Sm antigens were prepared Sltu in the laboratory of Dr. Eng Tan, Scripps Clinic and 0 Research Foundation, as previously described (30, 31). 0 Crystalline pancreatic DNase (2,460 U/mg) and RNase *= 50- I (3,095 U/mg) were purchased from Worthington Bio- chemical Corp. ['H]DNA from Escherichia coli, extracted L._ by modifications of the procedure of Lehman (32), was 0 supplied by Dr. Robert Department of E Swift, Microbiology 0I1 (Rochester). 28S [14C]RNA, phenol purified from rat liver, S 30- I 0 was the gift of Dr. Meisler. C. Experiments were undertaken to determine whether a-.! b treatment of lysosomes with high concentrations of DNase 0 or RNase would affect their capacity to give CF reactions 0 (series I) or precipitin reactions (series II) with SLE 10- sera. For this purpose the lysosomes were assumed to aflv __ 0' have DNA or RNA contamination equivalent to one-tenth age", Ura the lysosomal protein In series I, DNase was employed in 25 an enzyme: substrate (E: S) ratio of 1: 2 in the presence 5 5 25 5 25 5 25 of 0.01 M MgCl2, and incubation with lysosomes was at Lysosomal Protein-pg/ml 37'C for 60 min; RNase was employed in an E: S ratio FIGURE 2 Histogram showing range of CF reactions of at 2:1 at 50C for 60 min with gentle mixing. In series I SLE and normal sera against human leukocyte lysosomes concurrent control tubes containing added ['H]DNA or at concentrations of 5 and 25 ,ug protein/ml. Weaker re- ["'C]RNA, in addition to lysosomes and the appropriate actions obtained with lysosomes at 1 ug/ml and 125 Ag/ml nuclease (or diluent), permitted demonstration of effec- are not shown. tive cleavage of DNA or RNA under the conditions of the experiment (see Results). For this purpose the re- maining radioactivity precipitable in 10% TCA was trapped failed to give at least 10% corrected CF at any dilution on glass fiber filters and counted in a liquid scintillation were listed as negative in Table I. With each CF assay of counter, according to the general method of Meisler and unknown sera one or more known positive SLE sera and Tropp (33). In series II, treatment of lysosomes with the rabbit antilysosome serum were included as positive either DNase or RNase was performed at 370C at an controls. E: S ratio of 1:2 (for 60 min) or 1:10 (for 18 h), with Successful development of the above CF system depended 0.15 M MgC12 provided in the DNase studies. upon overcoming substantial anticomplementary activity of the lysosomes themselves and of many SLE sera. The Detection of antilysosome antibodies anticomplementary effect of lysosomes was circumvented by incorporating additional "carrier" protein in the system, Quantitative CF tests were patterned after the method i.e., the diluent was VBS containing 1:12 heat-inactivated of Wassermann and Levine (34). Sensitized sheep red normal human serum. Before CF assays, SLE sera were cells (EA), 5 X 101 cells/ml, in isotonic Veronal-buffered pretreated as follows: 3 ml 1:10 serum (in VBS) was saline (VBS) containing Ca+ and Mg" were prepared as incubated with 70 CH.o U of guinea pig complement at previously described (35). Only freshly prepared lyso- 370C for 60 min and then inactivated at 560C for 30 min. somes, stored no more than 3 days at 5'C, were used for This procedure' markedly reduced anticomplementary ac- these assays. Into 10-ml tubes in an ice bath were placed tivity of SLE sera but did not hinder positive CF reactions 0.5 ml diluent (see below), 0.25 ml diluted test serum, 0.25 with lysosomes. For uniformity, all human sera to be ml reconstituted lyophilized guinea pig serum containing 10 tested, including normals, were pretreated in this manner. CHra U/ml, and, lastly, 0.5 ml lysosomes diluted in 0.34 M Fractionation of 1-ml aliquots of selected SLE sera was sucrose, typically to S or 25 *g/ml. Antigen (lysosome), performed at 5°C in a 2.5 X 90-cm Sephadex G-200 column antibody (serum), and complement control tubes were run in borate-saline buffer, pH 8.0, with a flow rate of 8 in duplicate in each experiment The tubes were incubated ml/h. IgG-, IgA-, IgM-, and albumin-rich fractions were at 4VC for 18-20 h, then chilled in ice, and 0.25 ml of EA determined by immunodiffusion against specific antisera. were added to each tube. Incubation then proceeded at 370C Representative fractions were tested in CF assays against until the complement controls indicated 100%o lysis. The lysosomes (see below). tubes were centrifuged and the ODnnm of each supernate Precipitin analysis in gel. Tests for precipitating anti- was measured spectrophotometrically. The final percent of body against various antigens were made by double diffu- CF was calculated after subtracting the sum of any com- sion in glass petri dishes containing 25 ml freshly prepared plement consumption occurring in the antigen (lysosome) 0.4% agarose gel in phosphate-buffered saline, pH 7.3, control and serum control. In practice this total correction producing a gel thickness of 5 mm. Wells were 8 mm in seldom exceeded 4-5%, owing to the pretreatment described diameter and placed 4 mm apart. When intact lysosomes below. The CF values shown in the tables have all been corrected for these anticomplementary effects. Although 'Kindly suggested by Dr. Peter H. Schur, Robert Breck normal sera produced no detectable CF, SLE sera which Brigham Hospital, Boston, Mass. Lysosomal Antigen and Antibody in Lupus Erythematosus 259 TABLE I CF and Precipitin Reactions of SLE Sera with Human Leukocyte Lysosomes*

Percent CFt Gel precip- Serum dilution (reciprocal) itin reac- Patient 40 160 640 2,560 10,240 40,960 tion

1. Tay. 31 53 30 47 21 4 + 2. Tho. 78 63 NT§ NT 27 0 + 3. Mor. 68 53 50 NT NT NT + 4. Mem. 21 28 25 31 15 4 + 5. Kli. 8 29 24 31 12 1 + 6. Mai. 38 42 39 38 0 NT + 7. Mend. 22 48 52 7 NT NT + 8. Aik. 14 14 19 26 6 2 0 9. Cre. 8 17 23 14 NT NT 0 10. Del. 1 4 12 23 8 0 NT 11. Cia. 8 10 10 9 NT NT 0 12. Wel. 3 6 8 13 1 0 0 13. Dic. 5 4 7 12 3 2 0 14. Bob. 2 5 10 13 1 NT 0 15. Ciac. 0 5 9 15 8 3 NT 16. Fal. 0 0 8 16 4 3 NT 17. Mur. 1 8 13 17 7 0 NT 18. Cer. 1 10 12 14 8 4 NT 19. Bro. 10 12 9 4 NT NT 0 20. Van. 4 10 11 4 NT NT NT 21. Hes. 17 13 8 4 NT NT NT 22. Bla. 11 7 5 2 NT NT NT 23. Thom. 11 8 2 0 NT NT NT 14 other SLE 0-2 0-6 0-7 0-8 0-5 0 Oil Two lupoid hepatitis 2-3 6-8 11-13 16-21 1-8 0-2 NT 10 drug-SLE 0-2 0-2 0-2 0-1 NT NT NT 15 RA 0 0 0 0 NT NT NT Four polymyositis 0 0 0 0 NT NT NT 26 AHA¶ 0 0 0 0 0 0 NT Seven miscellaneous diseases** 0-6 0-1 0 0 NT NT NT 14 normals 0 0 0 0 0 0 0 * Lysosomes were tested at a protein concentration of 25 Iug/ml. 1 All values represent percent CF after correction for anticomplementary effects of lysosomes alone and of serum alone. § NT, not tested. Seven of the 14 patients were tested. ¶ Drug-SLE, drug-induced "lupus" syndrome; AHA, autoimmune hemolytic anemia. ** Miscellaneous rheumatic disorders including Wegener's granulomatosis (two cases), (two), Henoch-Sch6nlein purpura (one), chronic glomerulonephritis (one), and viral hepatitis with polyarthritis (one). were used as antigen, the well was completely filled at for study. Some of these patients had more limited forms least 6 h ahead of the serum wells and then refilled at the of SLE such as thrombocytopenic purpura with positive LE time of addition of serum. Lysosomes were used at a con- cell test and antinuclear antibody (ANA), or Coombs- centration of 1,000-1,500 ,ug/ml. Diffusion occurred during positive hemolytic anemia associated with strongly positive 24-48 h at room temperature. ANA, joint symptoms, and Raynaud's phenomenon. The rheumatoid arthritis (RA) patients included had definite Patients or classical RA. The procaine amide-induced lupus patients Sera from patients had been stored at -20'C before exhibited diagnostic features previously described (36). study. Pleural and joint fluids were examined, however, Cases listed as autoimmune hemolytic anemia did not have as fresh specimens. SLE cases were selected only on the clinical or laboratory features of SLE; most were idio- basis that clinical and laboratory evidence clearly sup- pathic and some were associated with lymphoproliferative ported the diagnosis and that sufficient serum was available disease. All of these patients were from the University of 260 D. A. Bell, P. A. Thiem, J. H. Vaughan, and J. P. Leddy Rochester Medical Center or Scripps Clinic and Research Foundation. Miscellaneous Rabbit antiserum to human lysosomes was prepared by two immunizations at 3-wk intervals with a total of 500 Ag of lysosomal protein in complete Freund's adjuvant in footpads and intramuscular sites. The antiserum used in E these studies was obtained 10 days after the last injection. 0C Purified rheumatoid factor (RF), generously provided by CM 0.4 -4 Dr. George Abraham, had been isolated from a patient with a Felty's syndrome with an exceptionally high latex titer 0 ~~~~~~~~-84 (1/100,000). After fractionation of the serum on Sephadex o w G-200, proteins eluting in the first peak were concentrated 0 "E and passed through another Sephadex G-200 column under 1.2 - 184ml t264ml *328ml 0 acid-dissociating conditions. The first contained potent z U peak 0 Patient Mem. IgMK RF exhibiting monoclonal properties in polyacryla- 1.0 5) mide gel electrophoresis and isoelectric focusing. 0z In SLE patients antibodies to native or single-strand DNA were measured by a Farr-type radioimmunoassay as 0. described (37) but with native or heat-denatured E. coli Cr~~~~~~~~~~~~~~~~~~~~~I ['H]DNA as antigens. Sl0.6 -1-16uo RESULTS 0.4- Immunologic reactions of SLE sera with 0.2 --8 lysosomes to eab e itain nSpae -0 ounI- CF tests. Lysosomes were tested at 1, 5, 25, and 125 b2520 30 t 35 40 45 50 Mg protein/ml.' CF proved to be most efficient at 5 ug/ml 256mp320m/184m1 and particularly at 25 ug/ml (Fig. 2), and with SLE FRACTION NUMBER serum dilutions in the 1: 40 to 1: 2,560 range (Table I). FIGuRE 3 Fractionation of antilysosome CF activity in Among the 37 SLE patients tested, serum from at least two SLE sera by gel filtration on Sephadex G-200 column. one date of bleeding in 23 patients (62%) gave positive Serial dilutions (1/2-1/16) of fractions designated by black CF reactions (Table I). CF reactions were negative at bars were tested against lysosomes at a concentration of 25 all dilutions tested with sera from 14 normal donors, 10 Ag protein/ml. patients with drug-induced lupus syndrome (due to pro- caine amide in nine, hydralazine in one), 15 patients CF reactions by IgG antilysosome antibodies and thus with RA (12 seropositive, three seronegative), four mask or reduce positive titers. When latex titers for RE patients with polymyositis, 26 patients with autoimmune were measured in all of the SLE sera listed in Table I, hemolytic anemia (not associated with SLE), and a no correlation was apparent between elevated latex titers small group of miscellaneous rheumatic disorders (Ta- and weak or absent antilysosome CE reactions. As a ble I, Fig. 2). Two cases of chronic active ("lupoid") more direct test of this issue, purified IgM RF (see hepatitis with positive ANA gave moderately positive Methods) was added to two latex-negative SLE sera: CF reactions with lysosomes (Table I). Tay.,5 possessing strong antilysosome reactivity and Aik., Rabbit antilysosome serum produced CF at com- exhibiting only moderate reactivity (see Table I). Con- parable lysosome and serum concentrations. Lysosomes trol aliquots received buffer alone. Latex titers were from six different group 0 donors gave positive CF raised to 1/5,120 in both RF-treated sera but there was reactions of generally similar strength with either rab- no change in CF reactions at any serum dilution, com- bit or SLE sera. Entirely comparable CF reactions were pared to the controls. obtained with lysosomes prepared by the alternate pro- Gel precipitin reactions. Seven of 21 SLE sera ex- cedure (see Methods). By gel filtration on Sephadex amined by immunodiffusion against lysosome prepara- G-200 the antilysosomal CF activity of two SLE sera tions demonstrated a single precipitin arc close to the eluted with IgG globulins (Fig. 3). antigen well (Table I). This reaction is illustrated in The theoretical possibility arose that the presence of Fig. 4 by serum Kli. A precipitin line against lysosome RF in certain SLE or RA sera might interfere with granules is clearly visible with undiluted serum and 'Lysosome concentrations indicated here and in the fig- faintly with 1: 4 serum. The precipitin line between ly- ures and tables are initial concentrations. Final concentra- sosomes and SLE serum Kli. was closer to the antigen tions in the CF reaction mixtures are 1/3 the values listed (see Methods). 6 Sera are identified by abbreviation of patient's name. Lysosomal Antigen and Antibody in Lupus Erythematosus 261 I). The faint precipitin lines between the rabbit anti- serum and the SLE sera (arrows, Fig. 4) are discussed below. Characterization of reactive lysosomal components. Multiple aliquots of lysosomes (25 ug/ml) were centri- fuged at 12,330-40,000 g for variable periods of time. Supernates and uncentrifuged lysosomes were compared in CF reactions with four SLE sera representing strong, moderate, and weak CF reactivity. As shown in Table II, a major loss of the original CF antigenic activity of the lysosomes with SLE sera was observed after cen- trifugation at 40,000 g for 3 h. Smaller losses of anti- genic activity were observed with shorter or slower centrifugations (serums Tay. and Kli.). Lysosomes at concentrations of 850 usg/ml or more (> 150 times the concentration capable of giving CF reactions with SLE serum) gave no reaction in the diphenylamine assay for DNA. This indicates that < 5 ,ug/ml of DNA is present in a typical undiluted lysosome preparation and < 0.15 og/ml in the dilute lysosome sus- pensions (25 ug/ml) which gave optimal CF reactions. Similarly, in two lysosome preparations containing 1,170 FIGURE 4 Immunodiffusion analysis (0.4% agarose) of SLE sera and rabbit antilysosome serum against human and 550 ug protein/ml, respectively, the measured (29) leukocyte lysosomes, 1.5 mg protein/ml (center well). Wells RNA content was 20 ug/ml. This implies an RNA con- 1 and 5: SLE serum Tay., undiluted and 1:4, respectively. tent of 0.017-0.036 ,ug/ml when such lysosome prepara- Wells 2 and 4: SLE serum Kli., undil. and 1: 4, respec- tions are diluted (25 ,ug/ml) for CF assays. tively. Wells 3 and 6: rabbit anti-lysosome serum, 1:4. Furthermore, as shown in Table III, the capacity to react in CF assays with SLE sera was not affected by (lysosome) well than the much stronger precipitin prior treatment of the lysosomes with high concentra- line(s) obtained with rabbit antilysosome serum (Fig. tions of DNase or RNase sufficient to cleave nearly all 4), suggesting that different antigenic components are of the added radioactive DNA or RNA serving as moni- detected by the SLE and rabbit immune sera by this tors of nuclease activity. method. SLE serum Tay. (9/68 bleeding) shows no Fig. 5 shows the results of gel precipitin reactions of reaction (Fig. 4), but an earlier pretreatment serum lysosomes and DNA with two different SLE sera, one of was clearly positive (see Table IV, below). Only those which (Tay., well 4) reacts with both DNA (well 3) SLE sera exhibiting the strongest CF reactions also and lysosomes (well 1). After extensive absorption with showed gel precipitin reactions with lysosomes (Table DNA, serum Tay. (well 5) no longer reacts with DNA

TABLE II Sedimentation of Lysosomal Antigens Reactive in CF with SLE Sera

Percent CF with SLE serum: Tay. Kui. Aik. Cer. Test antigen 1/640 1/1,280 1/2,560 1/640 1/2,560 1/64 1/2,560 1/640 1/2,560

Whole lysosomes* 35 38 45 25 31 20 25 11 16 SN$ (12,330 g, 60 min) -§ 26 17 SN (40,000 g, 20 min) - 17 13 SN (40,000 g, 60 min) 10 11 7 10 10 - - SN (40,000 g, 180 min) 6 6 3 4 2 5 5 4 3 * Uncentrifuged lysosome preparation. $ SN, supernate after centrifugation at g force indicated. I Not tested. 262 D. A. BeU, P. A. Thiem, J. H. Vaughan, and J. P. Leddy TABLE III Effect of DNase and RNase Treatment on Reactivity of Lysosomes with SLE Sera

Percent CF with :1

SLE serum Tay. SLE serum Mem. Rabbit antibody cpm remain- 1/320 1/1,280 1/320 1/1,280 Test material* Prior treatment ing (acid ppt)t 1/1,280

Lysosomes + [3H]DNA Diluentll 8,659 51 50 9 18 48 Lysosomes + [3H]DNA DNasell 126 49 48 10 17 47 Sucrose + [3H]DNA Diluentll 10,799 Lysosomes + [14C]RNA Diluent¶ 438 24 48 28 50 49 Lysosomes + [14C]RNA RNase¶ 19 28 47 29 49 48 Sucrose + [14C]RNA Diluent¶ 454 - - - * Lysosomes, 125 ;Ag + 5 mg['H]DNA or 6.4 pg [14C]RNA. I Radioactivity precipitable by 10% TCA after incubation (see Methods). § Lysosomes from prior step diluted to 25 ,g/ml for CF tests. 1 Incubation at 370C, 60 min, in presence of 0.01 M Mg++ with or without 20 jg DNase (2,460 U/mg). ¶ Incubation at 50C, 60 min, with or without 25 pg RNase (3,095 U/mg).

(well 6) but reacts just as strongly with lysosomes. The costeroid therapy or in association with a clinical re- second patient's serum(Mend., well 2) gives no visible lapse after steroids had been discontinued (patient reaction with DNA (well 3) but forms a precipitin arc Mem.). Among the other SLE patients, tested at a with lysosomes which fuses with that given by serum single point in time, many with negative or very low Tay. The precipitin line between wells 4 and 5 is at- tributable to excess DNA in the absorbed Tay. serum (well 5). In other studies (not shown) lysosomes and DNA, which had been incubated in parallel with DNase, were tested by immunodiffusion against SLE sera known to give precipitin reactions with both DNA and lysosomes. This treatment abolished the reactions of DNA with these SLE sera but did not affect the reactivity of ly- sosomes. Similarly, treatment of lysosomes with RNase had no effect on their precipitin reactions with SLE sera. SLE serum Mor. gave gel precipitin lines against both lysosomes and a partially purified preparation of the nuclear antigen, Sm (30). These appeared to be reactions of nonidentity but this was not conclusive be- cause of marked differences in the diffusion rates of these two antigens. Exhaustive absorption of Mor. serum with the Sm antigen, however, abolished precipitin reac- tivity with Sm but not with lysosomes, indicating that the precipitin reaction with lysosomes is not attributable to the Sm: anti-Sm system. Clinical correlations. Table IV summarizes retro- spective clinical data on a small group of SLE patients on whom the availability of multiple serum samples FIGuRE 5 Immunodiffusion reactions in 0.4% agarose with permitted serial measurements of antilysosome antibody. the following reactants: Well 1: human leukocyte lyso- somes, 1.5 mg protein/ml. Well 2: undiluted SLE serum In these cases potent antibody, as defined by CF titers Mend. Wells 3 and 6: native calf thymus DNA, 1 mg/ml. greater than 2,560 and positive precipitin reactions, was Wells 4 and 5: undiluted SLE serum Tay., unabsorbed found during periods of active disease prior to corti- (well 4) and absorbed with native DNA (well 5). Lysosomal Antigen and Antibody in Lupus Erythematosus 263 TABLE IV Serial Studies of Antilysosome Antibodies in Selected SLE Patients

Antilysosome antibody

CF reactions* Highest Precipitin Active ne- Peripheral Arthritis, Patient Date percent Titer reaction phritis serositis Therapy Tay. 1-9-67 53 10,240 + ++ + 0 None 9-11-68 11 640 0 0 0 0 Steroidsl 11-6-68 13 160 0 0 0 0 Steroidst Mor. 1-14-69 68 > 2,560 + ++ + NA§ None 1-20-69 17 2,560 0 ++ + NA Steroids 3-28-69 16 2,560 0 4 4 NA Steroids KM. 3-19-68 31 10,240 + ++ ++ ++ None 3-25-69 10 640 0 0 0 0 Steroids Mem. 8-28-68 16 2,560 0 0 0 0 Steroids 2-28-70 31 10,240 + + + NA None

* CF results are listed as highest percent CF at any serum dilution tested and as titer, i.e., highest serum dilution producing at least 10% CF. : Systemic , usually prednisone. § NA, data not available. titers of antilysosome antibody had been effectively cleoprotein, Sm antigen, or human immunoglobulins, all treated with steroids and were clinically inactive at the tested at concentrations similar to that of the lysosomes. time of study. On the other hand, there were several Studies were initiated to clarify the relationship of this patients in the series (on corticosteroids) who still antigenic component in SLE sera with lysosomes isolated had active SLE with nephritis, high anti-DNA levels, from normal donors. Lysosomes (1.5 mg/ml) prepared and depressed complement titers, with only modest or by the standard procedure were divided into two equal negative antilysosome reactions. A consistent correlation aliquots. One was subjected to 10 freeze-thaw cycles; between antibody to native or single-strand DNA and the other (control) aliquot was maintained at 370C. antilysosome antibodies was not found. Both preparations were centrifuged at 31,000 g (30 min) and the upper 2/3 supernate (SN) was carefully Detection of lysosomal antigen in serum and other body fluids A faint but reproducible precipitin line (arrows, Fig. 4) is visible between rabbit antilysosome serum (wells 3 and 6) and the two SLE sera (wells 4 and 5). A similar precipitin line with the rabbit antiserum was also ob- served with 10 of 17 other SLE sera, suggesting the possible presence of lysosomal antigen in these sera. Further studies established that such precipitin reac- tions (a) were not observed with normal human sera; (b) were not altered by heating the SLE serum to 560C for 30 min; (c) occurred with the 40 % ammonium sul- fate fraction of the rabbit antiserum, but not with preim- mune serum from the same animal nor with rabbit anti- sera to human gamma globulin or to denatured DNA; FIGuRE 6 Immunodiffusion reactions in 0.4% agarose with (d) were not affected by absorption of the rabbit anti- the following reactants: Well 1: SN after sedimentation normal human and were (31,000 g, 30 min) of intact granules. Well 2: intact, un- serum with pooled serum; (e) sedimented lysosome granules, 800 *g/ml. Well 3: freeze- not observed when rabbit antilysosome serum was dif- thaw granule SN after sedimentation (31,000 g, 30 min), fused against DNA (native and denatured), soluble nu- 475 1Ag/mI. Well 4: rabbit antilysosome serum, 1:4. 264 D. A. Bell, P. A. Thiem, J. H. Vaughan, and J. P. Leddy TABLE V CF Reactions Comparing Whole Lysosomes and a Soluble Frac- tion Obtained by Granule Disruption

Percent CF with:

Whole Soluble granule lyso- supernate* somes (25 jg/ 5 yg/ 25 ;pg/ 100 jug/ Antibody source ml) ml ml ml

SLE serum Tay. (1/640) 42 1 0 A-Ct Rabbit antilysosome FIGuix 7 Immunodiffusion reactions in 0.4% agarose with serum (1/1,280) 47 16 22 A-C the following reactants: Wells 1 and 3: granule supernates from two donors after freeze-thaw disruption and sedi- * Obtained by centrifugation at 31.000 g for 30 min after 10-cycle freeze- mentation at 31,000 g for 30 min. Well 2: SLE serum thaw treatment of freshly prepared whole lysosomes (see text). (Wei.), undiluted. Well 4: rabbit antilysosome serum, : The soluble fraction was excessively anticomplementary at this higher 1:8. concentration. aspirated. Immunodiffusion analysis (Fig. 6) revealed Another patient not included in Table VI had a transi- that a component antigenically identical to a component ent leukemoid reaction and myalgic syndrome of unde- in the whole ("intact") lysosomes6 was released into termined etiology. When sera were studied serially, ly- the granule SN by freezing and thawing. This was not sosomal antigen could be detected only while there was detectable in the control SN. In Fig. 7 the fusion of the a significant granulocytosis. In another patient, with precipitin lines given by the freeze-thaw granule SN SLE, lysosomal antigen appeared during some periods and by the lysosomal antigen in the SLE serum indi- of active disease but could not be correlated with the cates their close antigenic relationship. After chroma- level of granulocytosis. tography on Sephadex G-200 this antigenic activity of the granule SN was identified in the included volume DISCUSSION only and was not detectably depleted after centrifugation This study demonstrates that a significant number of pa- at 130,000 g for 4 h, suggesting that a soluble antigen tients with active SLE display CF and gel precipitin was involved. The same freeze-thaw granule SN, how- reactions with a sedimentable component of lysosome ever, showed no precipitin reactivity with three SLE preparations derived from normal human leukocytes. sera which had given precipitin reactions with intact In view of the wide array of autoantibodies character- lysosomes. As shown in Table V, even with the sensi- istic of SLE, considerable effort was made to evaluate tive CF assay there was no reaction between this freeze- the possibility that these reactions were actually due to thaw SN and a known CF-positive SLE serum; the rabbit antiserum, however, produced a clearly positive reaction, somewhat weaker than with whole lysosomes. TABLE VI These data provide further evidence that the rabbit Immunodiffusion Reactions of Rabbit Antilysosome Serum with antiserum and SLE sera react with different lysosomal Extracellular Fluids of Selected Patients constituents. No. positive Lysosomal antigen in various clinical disorders. The rabbit antilysosome serum was tested (in 0.4% agarose) Clinical disorder Fluid tested No. tested against sera and other extracellular fluids from patients SLE serum 10/17 with various clinical disorders and the results are sum- pleural 0/1* marized in Table VI. When present, these precipitin reactions other RA serum 7/10 lines always gave of identity with each synovial 6/61 (i.e., from one fluid to another or from case to case) pleural 1/11 and with whole lysosome preparations. Ankylosing spondylitis synovial 1/1§ O "Whole" or "intact" is used to denote lysosome prepara- tions which have not been deliberately disrupted by freeze- Normals serum 0/30 thaw treatment or by detergents. It is recognized, how- ever, that a portion of the total lysosomal enzyme content * Patient's serum was also negative. is detectable in fresh lysosome preparations, implying some Patients' sera were also positive. rupture of granules during isolation. § Serum was not available on this patient. Lysosomal Antigen and Antibody in Lupus Erythematosus 265 antibodies other than antilysosome antibodies reacting activity was demonstrated by these workers using anti- with one or more contaminating antigens in the lysosome gens at protein concentrations 12-20 times higher than preparations. Such a contaminating antigen would have in our (25 sg/ml) preparations. This might reflect the had to be present regularly and in substantial concen- presence of a relatively large proportion of nonlysosome tration to account for consistent gel precipitin reactions proteins in their admittedly crude (16) fractions, or a on the one hand and strong CF reactions with lysosomes relative inefficiency of heterologous antigens in the CF at concentrations as low as 5 ig/ml' on the other assay. (Fig. 2). The actual frequency of antilysosome antibodies in Several lines of evidence indicated that these immuno- SLE may not be accurately reflected by the data in Ta- logic reactions could not be attributed to DNA: anti- ble I and Fig. 2. In many of the present cases serum DNA or RNA: anti-RNA reactions. These include (a) from the most active or pretreatment phase of the illness the very low concentrations of DNA (< 5 isg/ml) and was not available. The limited data in Table III suggest RNA ( 20 ,ug/ml) in undiluted suspensions of ly- that therapy may effectively suppress such sosomes containing 550-1,500 ;&g protein/ml; (b) the antibody. failure of high concentrations of demonstrably active In addition to the observations on antilysosome anti- DNase or RNase to affect either CF or precipitin re- bodies, evidence has been presented for the occurrence of actions of lysosomes with SLE sera; (c) our ability to lysosomal antigen in serum, synovial fluid, and pleural absorb anti-DNA antibody from an SLE serum without fluid of patients with SLE or other inflammatory dis- affecting its reactivity with lysosomes; and (d) the lack eases. This lysosomal antigen, which was identified by of correlation of anti-DNA and antilysosome reactions gel precipitin reactions with rabbit antiserum to human in individual sera. leukocyte lysosomes, appeared to be antigenically identi- Autoantibodies to other subcellular cytoplasmic or- cal to a soluble component which can be released in vitro ganelles including mitochondria (8-11), microsomes from intact lysosomes of normal donors. Since this com- (9, 10), ribosomes (12-14), and soluble cytoplasmic ponent does not detectably react with SLE sera known antigens (15) have been identified by others in SLE to give precipitin or CF reactions with intact lysosomes, sera. It appears unlikely that the reactions of our SLE the naturally occurring lysosomal antigen appears to be sera with lysosomes are attributable to antimitochondrial either a distinct substance (e.g., a lysosomal enzyme antibodies because (a) mitochondrial structures were rather than membrane antigen) or a membrane antigen rare in our lysosome preparations by electron microscopy in a degraded form. (Fig. 1), (b) neither succinic dehydrogenase nor re- The appearance of this lysosomal antigen in a variety duced NAD dehydrogenase (cytochrome c reductase) of inflammatory fluids and in the serum of a non-SLE activities were detected, and (c) the sedimentation char- patient during a transient neutrophilic leukemoid reac- acteristics of our lysosomal antigen are not typical of tion is consistent with its origin from granulocytes mitochondria (38). Likewise, the RNase resistance and in vivo. Artifactual release of lysosomal antigen as a sedimentation properties of our lysosome antigen suggest result of cell damage during collection of blood or fluids that ribonucleoprotein, ribosomes, microsomes, or soluble with high granulocyte counts was also considered. While cytoplasmic constituents are not the antigens responsible this possibility cannot be dismissed, it seems unlikely in for the observed reactivity (see 9, 38, 39). Our inability view of the uniform absence of the antigen in normals to detect (Na', K+)-ATPase in lysosomal membranes is and its occurrence in SLE and RA patients with normal evidence against major contamination by fragments of or reduced peripheral blood granulocytes. It is probable the leukocyte plasma membrane (25). Although we that a variety of subcellular antigens may be released at cannot exclude the possibility of a nonlysosomal antigen sites of tissue injury of different etiologies (40). The bound to lysosome membranes, it seems reasonable to presence of DNA (41, 42) and of lysosomal enzyme ascribe the observed CF and precipitin reactions to the activities (43, 44) in pathologic joint fluids and of DNA presence of antibodies in the SLE sera reactive with in the serum of patients after corticosteroid therapy or membrane-bound antigen of lysosome granules. open heart surgery (42) are thought to be further ex- This study, utilizing extensively characterized prepara- amples of this process. Thus, the occurrence of soluble tions of human lysosomes, confirms and extends the lysosomal antigen in SLE serum (or other body fluids) previous reports of Wiedermann and associates on the may well prove to be a normal aspect of inflammation. occurrence of antilysosome antibodies in SLE (10, 16). The present investigations indicate, however, that The studies of these authors differ from ours in several what is abnormal in SLE is the formation of antily- respects. First, their published data are based largely on sosome antibody. This, in turn, raises the question of the CF reactions of SLE sera with cytoplasmic fractions possible significance of such antibody. Earlier studies from rat and guinea pig liver and kidney. Secondly, CF provided in vitro evidence for a protective effect of 266 D. A. Bell, P. A. Thiem, 1. H. Vaughan, and I. P. Leddy antilysosome antibodies, i.e., lysosome stabilization (16, and isolated glomeruli of kidneys from patients with 45). Wiedermann, Hirschhorn, Weissmann, and lupus nephritis. J. Clin. Invest. 46: 569-579. 4. Svec, K H., and S. T. Allen. 1970. Antibody to nu- Miescher (16), moreover, suggested that the autoanti- clear material eluted from isolated spleen vessels in body response to lysosomes may be a physiologic reac- systemic lupus erythematosus. Science (Wash. D. C.). tion to tissue injury. Yet, in the present studies, antibody 170: 550-551. to human lysosomes was not detected in sera of selected 5. Andres, G. A., L. Occinni, S. M. Beiser, C. L. Chris- groups of patients with a variety of other rheumatic or tian, G. A. Cinotti, B. F. Erlanger, K C. Hsu, and B. C. Seegal. 1970. Localization of fluorescein-labeled autoimmune disorders except lupoid hepatitis (Table I). antinucleoside antibodies in glomeruli of patients with To date, we have not demonstrated in SLE sera the active systemic lupus erythematosus nephritis. J. Clin. presence of the membrane-associated (sedimentable) Invest. 49: 2106-2118. lysosomal antigen with which the human antilysosome 6. Koffler, D., V. Agnello, R Thoburn, and H. G. Kunkel. 1971. Systemic lupus erythematosus: Prototype of im- antibodies react. Such material may remain in inflamed mune complex nephritis in man. J. Exp. Med. 134: 169s- tissues or be rapidly cleared from body fluids by phago- 179s. cytes. It is conceivable that in SLE patients possessing 7. Gajdusek, D. C. 1958. An "autoimmune" reaction against circulating antibody to lysosomes, leukocyte breakdown human tissue antigens in certain acute and chronic dis- eases. I. Serologic investigations. Arch. Intern. Med. at sites of inflammation could result in local formation 101: 9-29. and deposition of lysosome: antilysosome complexes with 8. Asherson, G. L. 1959. Antibodies against nuclear and potential for perpetuation of tissue injury. The relation- cytoplasmic cell constituents in systemic lupus erythe- ship, if any, of such antibodies in SLE patients to the matosus and other diseases. Br. J. Exp. Pathol. 40: other, soluble lysosomal antigen (i.e., that which is reac- 209-215. 9. Deicher, H. R. G., H. R. Holman, and H. G. Kunkel. tive with rabbit antiserum and antigenically similar to a 1960. Anti-cytoplasmic factors in the sera of patients nonsedimentable component released from intact gran- with systemic lupus erythematosus and certain other dis- ules) is not apparent at this time. eases. Arthritis Rheum. 3: 1-15. A final point of interest was the possible association of 10. Wiedermann, G., and P. A. Miescher. 1965. Cytoplasmic antibodies in patients with systemic lupus erythematosus. antilysosome antibody with leukopenia, thrombocyto- Ann. N. Y. Acad. Sci. 124: 807-815. penia, or Coombs-positive hemolytic anemia in SLE pa- 11. Doniach, D., I. M. Roitt, J. G. Walker, and S. Sher- tients. In vitro studies had suggested that the membranes lock. 1966. Tissue antibodies in primary biliary cirrhosis, of lysosomes, leukocytes, and erythrocytes share certain active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications. antigenic determinants (45-48) and that heterologous Clin. Exp. Immunol. 1: 237-262. antilysosome antibody may injure intact granulocytes 12. Sturgill, B. C., and R. R. Carpenter. 1965. Antibody to under experimental conditions (46). Retrospective analy- ribosomes in systemic lupus erythematosus. Arthritis sis of our patients' records did not demonstrate any of Rheum. 8: 213-218. these associations, in agreement with the conclusion of 13. Schur, P. H., L. A. Moroz, and H. G. Kunkel. 1967. Precipitating antibodies to ribosomes in the serum of Wiedermann and Miescher (10). patients with systemic lupus erythematosus. Immuno- ACKNOWLEDGMENTS chemistry. 4: 447453. 14. Lamon, E. W., and J. C. Bennett. 1970. Antibodies to This research was supported by training grant AI-00028 ribosomal ribonucleic acid (rRNA) in patients with and research grants AM-09810 and AM-15118 from the systemic lupus erythematosus. Immunology. 19: 439- U. S. Public Health Service. 442. In addition to the many personal acknowledgments in 15. Clark, G., M. Reichlin, and T. B. Tomasi, Jr. 1969. the text we wish to thank Dr. Arnold I. Meisler for his Characterization of a soluble cytoplasmic antigen re- helpful advice, Drs. C. Douglas Angevine and John J. active with sera from patients with SLE. J. Immunol. 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268 D. A. Bell, P. A. Thiem, J. H. Vaughan, and J. P. Leddy