Scand J Rheumatology 7: 241-246, 1978


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From the University of Toronto, Division of Experimental Pathology and the Rheumatic Disease Unit, The Wellesley Hospifal, Toronto, Ontario, Canada

ABSTRACT. The efficacy of soluble immune complexes (IC) of different sizes prepared in vitro or present in RA sera and synovial fluids to induce the release of 8glucuronidase (BG) and neutral protease (NP) from PMN has been examined. Immune complexes of human HGGrabbit anti-human HGG prepared in 5, 10 and 20 times excess of antigen equivalence were fractionated into three pools, PI (22S-l3S), PI1 (135-75) and PI11 (7s) using Sephadex 6-200 column chromatography. NP and BGreleasing activity was mostly associated with PII. Similar fractions were obtained from RA sera and synovial fluids. BG-releasing activity was again predominantly associated with PII. PI1 fractions from normal sera and from 2 nonRA IC disease sera showed less BG-releasing activity than the RA PI1 fractions. Negligible NP release was observed with all three serum pools. Further investigation demonstrated the presence of NP inhibitor(s) in P I and PI1 from human sera.

Considerable evidence exists which suggests that immunologic tissue injury is mediated by agents released from polymorphonuclear leukocytes (PMN’s) (1). Substances capable of degrading collagen, elastin, hyaluronic acid, protein polysaccharides of cartilage, etc. have been isolated from PMN’s ( 2 ) . It has been well documented that lysosomal constituents are released from PMN’s incubated with phagocytosable and non-phagocytosable immune complexes (3-6). It has been suggested that the inflammatory response in the rheumatoid arthritis (RA) synovium is due to activity of complexes of IgG with rheumatoid factor (7). Rheumatoid synovial tissue and synovial fluid can contain large amounts of intserstitial and intracellular IgG-complement deposits (8-10). Some synovial fluid complexes can react directly with phagocytic cells, while other may be phagocytosed after preincubation with IgM-RF ( I 1). Phagocytosis by these cells may lead to the release of lysosomal enzymes. In addition, in vitro experiments have shown that autologous IgG 16-78 I866

can react with IgG-RF to form complexes that will activate complement in synovial fluid (12). Intermediate size soluble immune complexes, sedimenting between 6.6s and 19S, have been isolated from serum and synovial fluid of patients with RA (12, 13). Subsequent investigation has shown that these complexes often contain IgG-RF and may be formed by preferential self-association of IgGRF (14). In vitro studies have demonstrated that either rheumatoid factor bound to aggregated IgG, or zymosan particles incubated with decomplemented serum from RA patients, can be phagocytosed by PMN and can induce lysosomal enzyme release ( IS, 16). However, little is known about the efficacy of soluble complexes from RA patients to interact with target cells. If self-associating IgG-RF or a specific lattice structure of immune reactants is involved in the synovitis of RA, then such complexes may be able to induce the release of mediators of inflammation from target cells. The present investigation was carried out to establish whether intermediate complexes from sera of patients with RA cause release of lysosomal enzymes from human PMN and to compare this release with the response obtained with isolated soluble immune complexes prepared in vim.

MATERIALS AND METHODS Isolation of neutrophils from human peripheral blood PMN were isolated from fresh peripheral blood of healthy volunteers according to the procedure of Steigbigel et al. (17). Blood containing 0.27% EDTA was diluted 1 : 2 with phosphate-buffered saline (0.1 M pH 7.4) and then 25 ml of this was layered over 10 ml of Ficoll-Hypaque solution (Hypaque from Winthrop Laboratories, Aurora, Ontario; Ficoll from Pharmacia, Uppsala, Sweden). After centrifugation at 400 g at 4°C for 20 minutes, the granuloScurid J Rherrmrito1oh.y 7


A . D . Morrison et al.

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cyte layer was isolated and mixed with 3% dextran (Dextran grade A from BDH Chemicals, Poole, England) to obtain a final concentration of 0.3% dextran and allowed to sediment for 90 minutes. The granulocyte layer was removed and centrifuged at 100 g, 4°C for 10 minuntes, and then mixed with 0.84% ammonium chloride in order to lyse the remaining erythrocytes. The cells were washed twice with Tyrode’s solution containing 0.1 % gelatin. The viability of cells was greater than 95% determined by trypan blue exclusion test. The cell suspension contained 80-9595 PMN. Preparution of pryformed immune complexes Antibodies to human gamma-globulin (HGG) were produced in male New Zealand white rabbits. The animals were immunized with subcutaneous injections of 1 mg Cohn Fraction I1 (Sigma Chemical Company, St. Louis, Mo.) emulsified in Freund’s complete adjuvant (Difco Laboratories, Detroit, Mich). The globulin fraction from the serum was isolated by sodium sulfate precipitation (18). IgM was removed by Sephadex G-200 (Pharmacia, Uppsala, Sweden) column chromatography equilibrated with 0.1 M Tris buffer (BDH Chemicals, Poole, England). pH 8.0 with 0.5 M NaCI. The IgG containing pool of the globulin fraction was concentrated on a Diaflo XM50 membrane (Amicon, Lexington, Mass.) and was equilibrated with 0.2 M sodium borate, pH 8.0. Specific antibody titers were estimated by quantitative precipitation reactions. Immune complexes were prepared at 5, 10, and 20 times excess of their antigen equivalence. The precipitated complexes were removed by centrifugation prior to application of the mixture to the Sephadex G-200 columns. The eluted materials were pooled into three fractions representing 22S-13S. 13S-7S, and 7s regions. These pools were concentrated on Diaflo XM-50 membranes and equilibrated in borate buffer. Sedimentation analysis of the pools was performed on a Beckman Spinco Model E analytical ultracentrifuge (samples were centrifuged at 52000 rpm, 50” phase plate angle or 60000 rpm, 60” phase plate angle, at 20°C in 0.2 M borate buffer). The concentration of protein in each pool was estimated from the optical density of the sample at 280 nm, assuming an extinction coefficient ( E : y m )of 14. Purification of serum sumples Serum (A-F) and synovial fluid (J,E,K) samples were obtained from 6 patients with rheumatoid arthritis, 1 patient with polymyositis having circulating IgG-anti-IgG complexes (P). and I patient with collagen disease with large amounts of intermediate immune complexes and hyperviscosity syndrome (H). Normal human serum ( I ) was also examined. The samples were fractioned by Sephadex G-200 column chromatography. The fractions were then tested for immunoglobulin content by immunodiffusion with appropriate antisera, and then pooled, concentrated and equilibrated in borate buffer according to the procedures used for the preparation of the complexes formed in v i m . Complexes from the nonrheumatoid sera (P,H) were obtained from Dr B. J. Underdown, Department of Medical Genetics. University of Toronto, Toronto, Ontario. The purification procedures

and complex size range in each pool were similar to those prepared in this laboratory. Large complexes (22S-13S) contained IgG, IgM, and IgA; intermediate complexes (13S-7S) contained mostly IgG and IgA and small amounts of IgM, and the 7 s fraction pool contained IgA and IgG (patient A had detectable amounts of low molecular weight IgM). The protein concentration in each pool was deter?ined from the optical density at 280 nm assuming an Ef;,,, of 14. Each pool was adjusted to a final concentration of 4 mg protein/ml. Incubation of PMN wirh immune complexes PMN (5x lo6 cells) in 0.5 ml Tyrode’s-gelatin solution were incubated with buffer alone or with 0.5 ml of the sample pool (4 mg) in a final volume of 1 ml at 37°C for 60 minutes in a metabolic shaker. The cells were removed by centrifugation and the supernatants were assayed for p-glucuronidase activity. Some of the supernatants were also examined for neutral protease activity. The viability of the cells after the reaction was estimated by trypan blue exclusion and, in some experiments, by determination of the release of the cytoplasmic marker lactic dehydrogenase (LDH). Enzyme ussays P-glucuronidase activity was assayed according to the method of Fishman (19) and LDH assays were performed by the method described by Bergmeyer et al. (20). Release was expressed as a percentage of the total enzyme present in a given number of cells. Total enzyme activity was determined by extraction of the cells with 0. I % Tnton X (Sigma Chemical Company, St. Louis., Mo.) after several freeze-thaw cycles. Neutral protease activity was determined by measurement of S35release from labelled rabbit ear cartilage as described by lgnarro et al. (21). Cartilage was labelled and prepared according to the procedure described by Ignarro et al. and then incubated with PMN supernatants at 37°C for 5 hours. After incubation, 0.2 ml aliquots were removed, added to 5 ml of Canamix (Canatech Inc., Toronto, Ontario) and analysed on a liquid scintillation spectrometer (Intertechnique). Total SS in the cartilage was determined by dissolving the cartilage in 2.0 ml of boiling concentrated HCL and then counting 0.2 ml aliquots in 5 ml of Canamix. The results were expressed as a percentage of the total S35(cpm) released.

RESULTS Response of P M N to preformed immune complexes Immune complexes (HGG-anti-HGG) prepared at 5, 10, and 20 times excess of antigen equivalence were separated into three pools, viz. I (22S-l3S), I1 (13S-7S), and 111 ( 7 9 , by column chromatography. Each pool was reacted in duplicate with human P M N ’ in ~ order to study the effect on lysosoma1 enzyme release. The relative amounts of enzymes by IC prepared in lox antigen excess are recorded in Fig. 1. It was found that the

Immune complex induced PMN-degrunulution

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Fig. 1. Sephadex (3-200 fractionation of soluble HGG-antiHGG complexes prepared in 10 times equivalence antigen excess. Relationship of complex size to the induction of

P-glucuronidase and neutral protease release from human PMN .

P-glucuronidase release induced by pools I , 11, and k 0.5%, 18.5% k 1.5% and 7.0% respectively. The PMN control in this experiment released 9.0%& 1.O% P-glucuronidase. Similar results were observed in four identical experiments. Neutral protease release induced by these fractions was 23%, 37%, and 18% respectively, while the PMN control released 23 %. The P-glucuronidase release induced by pools I , 11, and 111 from complexes prepared in 5 X antigen excess was 15.9% k 1.0%, 35.8% k 1.8%, 11.1% k 1.2% respectively. The PMN control released 8.0% k 0.1 %. In experiments using IC prepared in 20x antigen excess, P-glucuronidase release induced by pools I, 11, and 111 was 16.2%, 35.4% f 4.4%, and 14.3% f 0.2% respectively, while the PMN controls released 8.0% k 0.1%. LDH release was less than 10% in these experiments. 111, was 9.5%

Release of Pglucuroniduse from PMN by immune complexes from patients’ sera Sera from RA patients were separated into three pools: I (22S-l3S), I1 (13s-7s) and 111 (7s) by Sephadex G-200 column chromatography. The pattern of P-glucuronidase release from PMN which was induced by these fractions was similar to that observed with the soluble immune complexes prepared in vifro. However, the percentage release


induced by these fractions which contained the same amount of protein as the preformed complexes was often lower than that induced by the preformed complexes. The results of the experiments using serum from patient C are shown in Figure 2 . Pools I , 11, and I11 induced the release of 6.0%k1.0%, 12.5%+ 2.0%, and 7.0% P-glucuronidase, respectively. The amount of this enzyme released from PMNs reacted with complexes from pool I1 isolated from the sera of 5 other patients was found to be consistently higher than that from incubation with material from pools I or 111 (Table I). Samples from patients A and E were tested in two experiments, while sera from the other patients were tested only once. Non-rheumatoid arthritis associated complexes (patients P and H) were fractionated on Sephadex G-200 and separated into 3 pools. These samples were tested for their ability to induce 0-glucuronidase release from PMN (Table I). More enzyme was released from PI1 than from P I or PIII. However, the maximal release by this pool of 7.5% was significantly lower than that released by PI1 from the RA samples. The quantity of protein in pool I obtained from the serum of patient H was insufficient for analysis. ,Serum from patient P was tested 3 times. Normal hyman serum (I) separated on Sephadex (3-200 And pooled similarly to the RA pools was reacted with PMN on 4 separate occasions and examined for p-glucuronidase release. Release induced from PMN by P I1 was higher than from P I or P I11 but the difference was significantly lower than that induced by the RA complexes (Table I).








Fig. 2. Fractionation of serum from rheumatoid arthritis patient C on a Sephadex (3-200 column. Examination of type of immunoglobulin present in the fractions and the

release of P-glucuronidase (vertical bars) from human PMN by pooled fractions (7S, 7s-13s and 13s-22s). Scund J Rheiimoroloy?. 7


A . D . Morrison et al.

Table I. EfJect of patient serum (s) and synovidj7uid (sf, on the release of /%glucuroriidusefrom PMN X.D. = not determined p-glucuronidase release, k"

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Sephadex G-200 pool Patient




PMN blank

A (R.A.j s B (R.A.) s C (R.A.) s D (R.A.)s E (R.A.) s F (R.A.) s P (nan R. A , ) s H (non R.A.) 5 I (normal) s J (R.A.) sf E R.A.) sf K (R.A.) sf

7.8 f 0.3 13.8 t 0.0 6.0 f 1.0

15.0 2 1.2 19.1 f 0.7 12.5 f 1.5 16.3 f 1 . 1 48.2 f 5.1 21.0? 0.8 7.5 k 0.0 6.0 f 0.0 10.6 f 0.4 17.4 f 0.0 16.5 ? 0.6 79.0 k 16.8

6.5 k 0.5 7.8 f 0.7 7.0 f 0.0 8.1 f 1.2 1 I .9 f 0.5 8.3 f 0.3 4.4 2 0.3 6.0 ? 0.0 4.8 _+ 0.0 7.6 ? 0.0 9.3 ? 0.5 12.9 & 1.8

5.7 2 0.1 10.3 t 2.4 N.D. 10.3 f 2 . 4 12.6 k 0.4 5.3 t0.6 2.7 t 0.0 4.0 2 0.0 5.7 _+ 0.1 5.4 t 0.0 5.4 t 0.0 12.6 f 0.4

9.0 t 0.0 8.2 t 1.7 10.1 f 0.6

2.5 f 0.1 N.D. 8.9 f 0.4 11.8 _+ 0.4 7.2 i 0.3 1 1 . 1 f 1.4

/3-glucuronidase release from s a m p l e z ( s ) and E T n ; K ( s n a n d E (s); A (s) and I (s);B (s) and D (s);were determined during the same experiments.


IZflect of RA synovia1Juid on the release of Fglucuronidasefrom P M N Sephadex (3-200 column chromatography was used to separate RA synovial fluid samples E, J. and K , into 3 pools: 1 (22S-I3S),I1 (13s-7s) and 111 (7s). PI1 incubated with PMN induced more p-glucuronidase release than observed with PI or PI11 (Table I). Synovial fluid samples E and K were tested twice and sample J was tested once for /3glucuronidase releasing activity. Release of P M N neutral protease by serum immune complexes It was found earlier that performed immune complexes prepared in lox equivalence antigen excess released neutral protease from human PMNs. An attempt was made to establish whether immune complexes from RA sera would show similar effects. Sample pools 1, I1 and 111 obtained from G-200 fractionation of RA serum A . non-RA serum P and preformed IC prepared in lox equivalence antigen excess were reacted with PMN. It will be seen in Figure 3 that none of the fraction pools from the serum of the patient A (RA) and patient P (hyperviscosity syndrome) released NP. In fact the amount of NP release seen with these fractions was around 7%. which was less than the amount of N P released (20%) spontaneously from PMNs alone. The pool I1 of the preformed immune complexes on the other hand released 37% NP.

In order to investigate the reason for the lower NP values with serum fractions the following experiment was performed. Serum fractions P I , P I1 and PI11 of patient P containing 2 mg of protein were mixed with PI1 from performed complexes (5x antigen excess) containing 2 mg of protein and the this mixture was reacted with PMNs. Results of this experiment are reported in Figure 4. Pool 11 of preformed immune complexes released 58% NP. When this fraction was mixed with PI11 of patient's serum the release was 47.5%. On the other hand, the mixture of PI1 from preformed immune complexes with either PI or P 11 of the patient's serum reieased less than 5 % NP, which was substantially low when compared with spontaneous release.





E 0

a 210-



Antigen E x c e s s




Patient A miRheumataid A r t h m s ) #Pattent P I H y p e w s c O s i t y Syndrome)












0) 3



Fig. 3. Release of neutral protease by various fractions obtained from patient sera and preformed immune complexes. Pool I: 13S-22S; Pool 11: 7S-13s; Pool 111: 7s.

Immune complex induced PMN-degranulation

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Fig. 4. Demonstration of inhibitors of neutral protease in Sephadex G-200 fractionation pools of patient P serum.

These findings suggest that the P I and PI1 fractions of patient’s serum contain inhibitor(s) of neutral protease. DISCUSSION The investigation of the ability of preformed soluble immune complexes to induce release of the lysosoma1 enzyme, P-glucuronidase from human PMN showed maximum releasing activity associated with the fraction containing complexes of intermediate size (13s-7s). Complexes with a higher sedimentation coefficient induced little more BG release than the free IgG (7s pool). Similar fractions obtained from sera of patients with rheumatoid arthritis demonstrated the BG releasing activity mostly in pool I1 (13-7s) which contained intermediate complexes. Pool I1 (13s-yS) from the sera of nonrheumatoid patients could not activate as much enzyme release. This may be due either to a low concentration of a particular size complex, or to the fact that the nature of these serum complexes is such that they are incapable of interacting with PMNs. Ignarro et al. (15) could not demonstrate lysosomal enzyme release from PMNs incubated with rheumatoid sera. However, they observed the release of BG from PMNs incubated with zymosan particles treated with decomplemented rheumatoid sera. The inability of unfractionated RA sera to release lysosomal enzymes may be due to the inhibitory effect of free IgG in the sera. This is in


agreement with an earlier observation that free IgG can compete for Fc receptors on the cell surface, thereby inhibiting the interaction of complexes with the cells (22). Although the present investigation showed that preformed intermediate complexes containing IgG-anti IgG and the intermediate complexes from sera and synovial fluid of RA patients released lysosomal enzymes, there are no substantial data available to suggest that it is the size of the complexes that determines the ability to induce lysosomal enzyme release. It should be noted that the nature of the complexes in pool I1 has not been completely determined. Pool I1 of the preformed complexes containing 2 mg of protein released more BG than the same amount of protein from pool I1 of most RA serum and synovial fluid samples. However, it should be taken into consideration that 2 mg of pool 11 from RA serum contains a number of other proteins in addition to the complexes. Therefore, it is essential to determine the amount of protein associated with the complexes in these pools. Neutral protease from human PMNs is capable of degrading proteoglycans and thus may play a role in the destruction of the joint in RA (21). It was observed that preformed intermediate immune complexes can induce the release of this enzyme from PMN. Serum proteins such as a,-antitrypsin and a,-macroglobulin are known to inhibit NP activity (23). Janoff et al. (24) have described NP inhibitor(s) present in the cytosol fraction of PMN having a molecular weight greater than 300000. Our studies on serum fractionated on Sephadex G-200 have demonstrated NP inhibitors in the large (22s-13s) and intermediate (13s-7s) size pools. It has been suggested that the effects of these inhibitors may be reduced in vivo when numerous PMN in the arthritic joint react with immune complexes bound to or near the articular surface, thus releasing NP into the vicinity of the cartilage, and thereby bringing about local damage (25). These studies suggest that the soluble immune complexes of intermediate size (13s-7s) are more efficient in inducing lysosomal enzyme release from human PMN than larger soluble complexes. Studies also indicate that the intermediate size complexes in sera from RA patients are capable of releasing lysosomal enzymes when they are separated from free IgG. Further characterization of these complexes is in progress. Scand J Rheumaiofogi 7


A . D . Morrison et al. ACKNOWLEDGEMENT

This work was supported by the Medical Research Council of Canada MT-3401.

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REFERENCES I . Cochrane, C. G.: Immunological tissue injury mediated by neutrophilic leukocytes. Adv Immunol Y;97, 1968 2 . Goldstein. I . M. & Weissman. G . : Cellular and humoral mechanisms in immune complex injury. I n Progress in Immunology. 11 ( 5 ) (ed. L. Brent and J. Holborow), pp. 81-90. North-Holland Publ. Co., Amsterdam. 1974. 3. Henson, P. M.: The immunologic release of constituents from neutrophil leukocytes. I . The role of antibody and complement on non-phagocytosable surfaces or phagocytosable particles. J Immunol 107: 1535, 1971. 4. Taichman, N . S . . Pruzanski, W. & Ranadive, N . S.: Release of intracellular constituents from rabbit polymorphonuclear leukocytes exposed to soluble and insoluble immune complexes. Int Arch Allergy 43: 182. 1972. 5 . Ranadive. N . S . . Sajnani. A . N.. Alimurka. I(. & Movat, H. Z.: Release of basic proteins and lysosomal enzymes from neutrophil leukocytes of the rabbit. Int Arch Allergy 45: 880, 1973. 6 . Movat, H. 2..Macmorine. D. R. L., Takeuchi. Y. & Burrowes, C. E.: Chemical mediators released by PMN-leukocytes during phagocytosis of Ag-Ab complexes. I n Cellular and Humoral Mechanisms in Anaphylaxis and Allergy (ed. H. 2. Movat). pp. 164-175. Kirrger, Base1 and New York, 1%9. 7. Rheumatoid Arthritis: I n Primer on the Rheumatic h s e a s e s led. G . P. Rodnan). JAMA 224. No 5 (Suppi.). 687-700, 1973. 8. Kinsella, T. D., Baum. J. Ziff, M.: fmmunofluorescent demonstration of an IgG-P,c complex in synovial lining cells of rheumatoid synovial membrane. Clin Exp lmmunol4: 265, 1969. 9. Rawson, A. J.. Abelson. N. M. & Hollander, J. L.: Studies on the pathogenesis of rheumatoid joint inflammation. 11. Intracytoplasmic particulate complexes in synovial fluids. Ann Int Med62: 281, 1%5. 10. Natvig. J. B. & Munthe, E.: Self-associating IgG rheumatoid factor represents a major response of plasma cells in rheumatoid inflammatory tissue. Ann NY Acad Sci 256: 88, 1975. I I . Hurd, E. R.. LoSpalluto, J. & Ziff, M.: Formation of leukocyte inclusions in normal polymorphonuclear cells incubated with synovial fluid. Arhritis Rheum 13:724. 1970 12. Winchester, R. J.. Agnello, V. & Kunkel, H. G.: Gamma-globulin complexes in synovial fluid of patients with rheumatoid arthritis. Partial characterization and relationship to lowered complement levels. Clin Exp Immunol6: 689, 1970 13. Kunkel, H . G . , Muller-Eberhard. H. J.. Fudenberg, H. H. & Tomasi. T. B.: Gamma-globulin complexes

in rheumatoid arthritis and certain other conditions. J Clin Invest40: 117, 1961. 14. Pope, R. M., Teller, D. C. & Mannik, M.: The molecular basis of self-association of antibodies to IgG (rheumatoid factors) in rheumatoid arthritis. Proc Nat Acad Sci (USA) 71: 517, 1974. 15. Ignarro, L. J., Lint, T. F. George, W . J.: Hormonal control of lysosomal enzyme release from human neutrophils. Effects of autonomic agents on enzyme release phagocytoses and cyclic nucleotide levels. J Exp Med 139: 1295, 1974. 16. Weissmann, G . , Zurier, R. B., Spieler, P. J. &Goldstein, I . M.: Mechanisms of lysosomal enzyme release from leukocytes exposed to immune complexes and other particles. J Exp Med 134: 149s, 1971. 17. Steigbigel, R. T., Lambert, L. H. & Remington, J. S.: Phagocytic and bactericidal properties of normal human monocytes. J Clin lnvest53: 131, 1974. 18. Marrack, J. R., Hoch, H. & Johns, R. G. S.: The valency of antibodies. Br J Exp Patho132:212, 1951. 19. Fishman, W. H.: P-glucuronidase. i n Methods of Enzymatic Analysis (ed. Bergmeyer), p. 929-943. Academic Press New York, 1974. 20. Bergmeyer. H. U.: Lactate dehydrogenase. I n Methods of Enzymatic Analysis (ed. Bergmeyer), p. 574. Academic Press New York, 1974. 21. Ignarro, L. J., Oronsky, A. L. & Perper, R . J.: Breakdown of non-collagenous chondromucoprotein matrix by leukocyte lysosomes granule lysates from guinea pig, rabbit, and human. Clin Immunol Immunopathol2: 36. 1973. 2 2 . Sajnani, A. N., Ranadive, N . S. & Movat, H. 2 . : Redistribution of immunoglobulin receptors on human neutrophils and its relationship to the release of lysosomal enzymes. Lab Invest35: 143, 1976. 23. Ohlsson, K. & Olsson, I.: Neutral proteases of human granulacytes. 111. Interaction between human granulocyte elastase and plasma protein inhibitors. Scand J Clin Lab Invest34: 349, 1974. 24. Janoff, A., Blondin, J., Sandhaus, R. A. Mosser, A. & Malemud, C.: Human neutrophil elastase: In vitro effects on natural substrates suggest important physiological and pathological actions. In Proteases and Biological Control (ed. E. Reich, D. B. S. Riffein and E. Shaver), Cold Spring Harbor Conferences on Cell Proliferation 2: 603, 1975. 25. Janoff, A., Feinstein, G . , Malemud, C. J. & Elias, J. M.: Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases. A model of joint injury. J. Clin Invest 57:615, 1976.

Submirted f o r publication February 6, 1978

Dr N. S. Ranadive Room 63 16 Division of Experimental Pathology Medical Sciences Building University of Toronto Toronto, Ontario, Canada M5S IA8

Release of lysosomal enzymes from human polymorphonuclear leukocytes by soluble intermediate immune complexes.

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