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We found that short-term culture medium and homogenate of casein-induced rat peritoneal polymorphonuclear leukocytes (PMN) markedly induced collagenase and prostaglandin E2 (PGE2) production by normal rat synovial cells and these effects were abrogated by anti-(rat interleukin-1 alpha) (IL-1 alpha) polyclonal antibodies. However, collagenase activity and PGE2 induced by recombinant rat IL-1 alpha were less than those induced by rat PMN culture medium. It was also proved by radioimmunoassay that rat PMN culture medium contains a relatively small amount of IL-1 alpha. The introduction of IL-1 alpha-deleted PMN culture medium and recombinant rat IL-1 alpha together into the synovial cell culture system revealed that IL-1 alpha deleted PMN culture medium has a significant enhancing activity on IL-1 alpha-induced synovial cell collagenase and PGE2 production. This new factor, which was shown to be a negatively charged protein of about 80 kDa, may have important roles in connective tissue destruction and chronic inflammation in diseases such as rheumatoid arthritis.

The fusion of lysosomes and other granules with phagocytic vesicles (endo-fusion) and cell membrane (exocytosis) was simultaneously examined in non-phagocytosing guinea-pig polymorphonuclear leukocytes (PMNs). beta-Glucuronidase as a typical lysosomal enzyme, acid phosphatase as another lysosomal enzyme, and alkaline phosphatase as a specific granule enzyme were assayed. PMNs released these three enzymes in the presence of serum but the extents of exocytosis differed considerably: release of beta-glucuronidase, alkaline phosphatase and acid phosphatase was 6.9, 4.3 and 3.3%, respectively. Acid phosphatase was released even in the absence of serum, whereas the other two enzymes were not. These three enzymes showed also different responses in endofusion: the preformed phagocytic vesicles fused with the granules containing beta-glucuronidase and acid phosphatase, but scarcely fused with those containing alkaline phosphatase, although all these enzymes were recovered in increasing amounts in the phagolysosome fraction when the cells were allowed to phagocytose continuously. These results suggest that fusion of these three types of granules with phagocytic vesicles (endo-fusion) and plasma membrane (exocytosis) is heterogeneous and regulated by different mechanisms in guinea-pig PMNs.

Stimulation of synovial cell prostaglandin production by a factor obtained from casein-induced peritoneal polymorphonuclear (PMN) cells has been investigated. Both the extract and short time cultured medium of rat peritoneal PMN cells stimulate prostaglandin (PG)E2 production as well as collagenase production in the culture of rat synovial cells. PGE2 production by the cells in the presence of the PMN factor is much faster (5 to 24 hr) than collagenase production (24 hr or later, Biomedical Res. 3, 506-516, 1982). This stimulating factor is confirmed to be derived from PMN cells, based on the purification of the cells from peritoneal exudate cells by the Ficoll-Urographin method. Elution profile of the factor on gel filtration has indicated that both PGE2 and collagenase productions by synovial cells are stimulated by the same effluent fractions corresponding to molecular weights of 15,000 - 20,000 daltons and 30,000 - 40,000 daltons. These results suggest that PMN cells are involved in PG production as well as collagenase production in the inflamed tissue by stimulating connective tissue cells such as synovial cells.

The rates and extents of fusion between lysosomes and phagocytic vesicles containing different kinds of particles were investigated kinetically in guinea pig polymorphonuclear leukocytes (PMNs). Particles were prepared by emulsifying paraffin oil with bovine serum albumin (POE-BSA), with hemoglobin (POE-Hb), and with ovalbumin (POE-OA). PMNs phagocytosed these particle in the order, POE-BSA greater than POE-Hb greater than POE-OA. As for fusion with lysosomes, the recoveries of lysosomal enzymes in the phagolysosome fraction during phagocytosis did not vary greatly, but the ratios of lysosomal enzymes to paraffin oil in the phagolysosome fractions, designated as "the fusion index," showed considerable differences, varying in reverse order to that of phagocytosis. Further studies were carried out on fusion between lysosomes and phagocytic vesicles as distinct from phagocytosis. PMNs were allowed to phagocytize particles at 37 degrees C for 5 min, then were washed free from non-ingested particles and reincubated without phagocytizable particles. The fusion index showed a slow but continuous increase in cells with POE-BSA, and a rapid but temporary increase in those with POE-Hb and POE-OA at 37 degrees C. These results suggest that different particles caused differences in the rate and extent not ony of phagocytosis but also of fusion with lysosomes.