The Histochemical Journal

, Volume 6, Issue 4, pp 431–446 | Cite as

The histochemical demonstration of hydrolytic enzymes in adjuvant-induced arthritis in rats

  • A. D. Pearse


Acid phosphatase, alkaline phosphatase, leucine naphthylamidase and β-glucuronidase were studied histochemically in unfixed, undemineralized affected paws from rats with adjuvant-induced arthritis. Sections were prepared in a newly designed bone cryostat and it was found that untreated tissue sections gave the most satisfactory results. Acid and alkaline phosphatase activities were studied using azo dye techniques and the substrate naphthol AS-TR phosphate gave the best results with regard to sensitivity and localization.

Parallel to the pathological changes in the rats, alkaline and acid phosphatase activities increased with noticeable changes in localization in degraded zones. The degradation of the articular cartilage was accompanied by an extracellular particulate reaction for acid phosphatase around the chondrocytes. The increase in β-glucuronidase activity was confined to its usual localization in synovial and inflammatory cells. Leucine naphthylamidase activity increased together with a change of the localization in the connective tissue of the affected joints.

The results suggest that acid phosphatase, alkaline phosphatase and leucine naphthylamidase play an active role in cartilage and bone erosion in adjuvant arthritis, whereas β-glucuronidase remains localized in inflammatory and synovial cells and does not appear to participate actively in the destructive process.


Alkaline Phosphatase Articular Cartilage Phosphatase Activity Acid Phosphatase Alkaline Phosphatase Activity 
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  1. Ali, S. Y. &Evans, L. (1969). Studies in the cathepsins in elastic cartilage.Biochem. J. 112, 247–433.Google Scholar
  2. Ali, S. Y., Evans, L., Stainthorpe, E. &Lack, C. H. (1967) Characterisation of cathepsins in cartilage.Biochem. J. 105, 549–57.Google Scholar
  3. Anderson, A. J. (1970). Lysosomal enzyme activity in rats with adjuvant-induced arthritis.Ann. Rheum. Dis. 29, 307–13.Google Scholar
  4. Anderson, H. C., Matsuzawa, T., Sajdera, S. W. &Ali, S. Y. (1970). Membranous particles in calcifying cartilage matrix.Trans. N. Y. Acad. Sci. 32, 619–30.Google Scholar
  5. Balough, K., Jr. (1962). Decalcification with versene for histochemical study of oxidative enzyme systems.J. Histochem. Cytochem. 10, 232–3.Google Scholar
  6. Barka, T. &Anderson, P. J. (1965).Histochemistry, Theory, Practice and Bibliography, p. 231. New York, Evanston & London: Holber Medical Division, Harper and Row.Google Scholar
  7. Barland, P., Novikoff, A. B. &Hammerman, D. (1964). Lysosomes in the synovial membrane in rheumatoid arthritis: a mechanism for cartilage erosion.Trans. Ass. Am. Phycns. 77, 239–47.Google Scholar
  8. Barrett, A. J. (1970) The role of lysosomes in arthritis.Adv. Sci. 27, 140–3.Google Scholar
  9. Barrett, A. J. &Poole, A. R. (1969). Unsuitability of Leucine Naphthylamide for the Histochemical Demonstration of Lysosomal Proteolytic Activity.Nature, (Lond.) 224, 279–80.Google Scholar
  10. Bertelli, A. (1968). Proteases and antiproteases substances in the inflammatory response.Biochem. Pharmacol. Suppl. 229, 40.Google Scholar
  11. Brandes, D. &Anton, E. (1969). Lysosomes in uterine involution: intracytoplasmic degradation of myofilaments and collagen.J. Geront. 24, 55–68.Google Scholar
  12. Burstein, N. A. &Waksman, B. H. (1964). The pathogenesis of adjuvant disease in the rat. I. A histologic study of early lesions in the joints and skin.Yale J. biol. Med 37, 177–94.Google Scholar
  13. Burstone, M.S. (1959). Histochemical demonstration of acid phosphatase activity in osteoclasts.J. Histochem. Cytochem. 7, 39–41.Google Scholar
  14. Chayen, J., Bitensky, L., Butcher, R. G. &Poulter, L. W. (1969).A Guide to Practical Histochemistry, p. 161. Edinburgh, Oliver and Boyd.Google Scholar
  15. Chayen, J. &Bitensky, L. (1971). Occasional survey: Lysosomal enzymes and inflammation, with particular reference to rheumatoid diseases.Ann. Rheum. Dis. 30, 522–36.Google Scholar
  16. Dingle, J. T. (1969). The extracellular secretion of lysosomal enzymes. In:Lysosomes in Biology and Pathology (eds. J. T. Dingle and H. B. Fell) Vol. 2, pp. 421–34. Amsterdam & London: North-Holland.Google Scholar
  17. Dingle, J. T. (1971). The immunoinhibition of cathespin D mediated cartilage degradation. In:Tissue Proteinases (eds. A. J. Barrett and J. T. Dingle), p. 313. Amsterdam, North-Holland.Google Scholar
  18. Duve, C. de (1969). The lysosome concept in retrospect. In:Lysosomes in Biology and Pathology (eds. J. T. Dingle and H. B. Fell), Vol. 1, pp. 3–40. Amsterdam & London: North-Holland.Google Scholar
  19. Fullmer, H. M. &Link, C. C. Jr (1964). A demineralization procedure for enzymatic histochemical use: A quantitative succinic dehydrogenase assay.Stain Technol. 39, 387–96.Google Scholar
  20. Glenn, E. M. (1966). Adjuvant induced arthritis: Effects of certain drugs on incidence and clinical severity, biochemical changes. Am. J. vet. Res.27, 339–52.Google Scholar
  21. Glenn, E. M., Gray, J. &Kooyers, W. (1965). Chemical changes in adjuvant-induced polyarthritis in rats.Am. J. vet. Res. 26, 1195–203.Google Scholar
  22. Hayashi, M., Nakajima, Y. &Fishman, W. H. (1964). The cytologic demonstration of β-glucuronidase employing naphthol AS-Bi glucuronide and hexazonium pararosanilm; a preliminary report.J. Histochem. Cytochem. 12, 293–7.Google Scholar
  23. Jones, R. S. &Ward, J. R. (1966). Adjuvant-induced polyarthritis in rats. In:Methods and Achievements in Experimental Pathology (eds. E. Bajusz and G. Jasmin), Vol. 1, pp. 607–38. Basel & New York: Karger.Google Scholar
  24. Kalliomaki, J. L. &Vanha-Perttula T. (1972), Activity of four hydrolytic enzymes in serum and synovial fluid of patients with rheumatoid arthritis.Scand. J. Rheum. 1, 21–6.Google Scholar
  25. Lowe, J. T. (1964). Serum protein changes in rats with arthritis induced by my cobacterial adjuvant.Biochem. Pharm. 13, 633–41.Google Scholar
  26. McDonald, J. K., Callahan, P. X., Ellis, S. &Smith, R. E (1971). Polypeptide degradation by dipeptidyl aminopeptidase I (cathespin C) and related peptidases. In:Tissue Proteinases (eds. A. J. Barrett and J. T. Dingle) p. 69. Amsterdam: North-Holland.Google Scholar
  27. Nachlas, M. M., Crawford, D. T. &Seligman, A. M (1957). The histochemical demonstration of leucine aminopeptidase.J. Histochem. Cytochem. 5, 264–78.Google Scholar
  28. Page Thomas, D. P. (1969). Lysosomal enzymes in experimental and rheumatoid arthritis. In:Lysosomes in Biology and Pathology (eds. J. T. Dingle and H. B. Fell), Vol. 2, pp. 87–106. Amsterdam & London: North-Holland.Google Scholar
  29. Pearse, A. D. (1973) The sharpening of Jung K microtome knives with particular reference to cryostat sectioning.J. med. Lab. Tech. 30, 117–22.Google Scholar
  30. Pearse, A. D. &Gardner, D. L. (1972). Preparation of unfixed undemineralized bone sections: The Bright bone cryostat.J. Clin. Path. 25, 26–9.Google Scholar
  31. Pearson, C. M. (1956). Development of arthritis, periarthritis, and periostitis in rats given adjuvants.Proc. Soc. exp. Biol. Med. USA 91, 95–101.Google Scholar
  32. Pearson, C. M., Waksman, B. H., &Sharp, J. T. (1961). Studies of arthritis and other lesions induced in rats by injection of mycobacterial adjuvant. V. Changes affecting the skin and mucous membranes. Comparison of the experimental process with human diseases.J. exp. Med. 113, 485–509.Google Scholar
  33. Smith, C. &Hammerman, D. (1962). Acid phosphatase in human synovial fluid.Arthr. Rheum. 5, 411–14.Google Scholar
  34. Stoerk, H. C., Bielinski, T. C. &Budzilovich, T. (1954) Chronic polyarthritis in rats injected with spleen in adjuvants.Am. J. Path. 30, 616.Google Scholar
  35. Vainio, U. (1970). Leucineaminopeptidase in rheumatoid arthritis, localization in sub-chondral bone and in synovial fluid cells.Ann. Rheum. Dis. 29, 434–8.Google Scholar
  36. Weimer, H. E., Wood, F. D. &Pearson, C. M. (1968). Serum protein alterations in adjuvant-induced arthritis.Canad. J. Biochem.,46, 743–8.Google Scholar
  37. Weissmann, G., Becher, B., Weidermann, G. &Bernheimer, A. W. (1965). Studies on Lysosomes. VII. Acute and chronic arthritis produced by intra-articular injections of streptolysin S in rabbits.Am. J. Pathol. 46, 129–47.Google Scholar
  38. Wergedal, J. E. &Baylink, D. J. (1969) Distribution of acid and alkaline phosphatase activity in undemineralized sections of the rat tibial diaphysis.J. Histochem. Cytochem. 17, 799–806.Google Scholar

Copyright information

© Chapman and Hall Ltd 1974

Authors and Affiliations

  • A. D. Pearse
    • 1
  1. 1.Experimental Pathology DivisionThe Kennedy Institute of RheumatologyLondonUK

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