In vitro effect of shark cartilage on human leukocyte function

OF THE THESIS IN VITRO EFFECT OF SHARK CARTILAGE ON HUMAN LEUKOCYTE FUNCTION by Aline Comelissen Florida International University, 2001 Miami, Florida Professor Sylvia L. Smith, Major Professor Previous in vitro studies have shown that shark cartilage extracts stimulate human leukocytes to release significant levels of TNFa, a cytokine typical of a Thl immune response. The purpose of this study was to investigate further the effects of shark cartilage on cellular immune function, particularly cell proliferation, apoptosis, and IL-4 and INFy production. The viability and proliferation of cell cultures grown in the presence of shark cartilage extract was not significantly different from unstimulated control cultures or those stimulated with mitogens (Con A, PMA, LPS), respectively. The effect of shark cartilage on apoptosis was determined by microscopic analysis of morphological apoptotic changes and by the detection of DNA fragmentation observed as characteristic ladder formation in agarose gel electrophoresis. While DNA fragmentation could not be demonstrated for cartilage-stimulated cells, characteristic morphological changes, indicative of apoptosis, were observed in leukocytes following incubation with shark cartilage extract. A statistically significant difference was not noted in the number of apoptotic cells present in cartilage-stimulated leukocytes and those stimulated with 0.5 pM/ml of staurosporine, suggesting that apoptosis is induced in the presence of cartilage extract. Culture supernatants of cartilage-stimulated leukocytes were assayed for IL-4 and IFN-y by enzyme-linked immunosorbent assay (ELISA). Although a low level of IL-4 and INFy was detected in culture supernatants of Con A and PMA stimulated cells it was not significantly different from that of unstimulated control cultures. Thus the significance of the absence of detectable IL-4 or INFy in supernatants of cartilagestimulated cultures could not be determined. However, as previously shown, supernatants did contain TNFa. Results of the study did not show a definitive pattern of cytokine production, characteristic of either a Thl or Th2 type immune response. Introduction............................................................................................................................... 1 Background...............................................................................................................................3 Material and Methods............................................................................................................24 Blood Donors..................................................................................................................... 24 Chemical and Reagents......................................................................................................24 Sterilization........................................................................................................................ 24 Cell Culture Medium..........................................................................................................24 Shark Cartilage Extract......................................................................................................24 Protein Determination........................................................................................................25 Assay for Endotoxin...........................................................................................................26 Isolation of Human Peripheral Blood Leukocytes...........................................................28 Protocol A ...................................................................................................................... 28 Protocol B ....................................................................................................................... 29 Protocol C ....................................................................................................................... 29 Cell Viability...................................................................................................................... 30 Leukocyte Cultures............................................................................................................31 Cell Proliferation...........................................................................................................31 MTT Assay..........................................................................................................................32 Leukocyte Culture for Cytokine Induction...................................................................... 33 Leukocyte Culture for Assessment of Apoptosis.........................................................34 Preparation of Leukocyte Cytospin monolayers.............................................................. 35 Assay for Extracellular Cytokine in Stimulated Cell Cultures....................................... 37 TNF-a Assay.................................................................................................................. 37 IFN-y Assay.....................................................................................................................38 IL-4 Assay...........................................................................................................................39 Isolation of Leukocyte DNA............................................................................................. 40 Electrophoretic Analysis of D NA .....................................................................................41 Statistical Analysis............................................................................................................. 42 Results......................................................................................................................................43 Shark Cartilage Extract......................................................................................................43 Endotoxin A ssay................................................................................................................ 43 Comparison of Cell Separation Methods..........................................................................45 Viability and Proliferation of Leukocyte in culture.........................................................46 Morphology of Cultured Leukocytes................................................................................ 52 Effect of Shark Cartilage on in vitro Leukocyte Proliferation........................................ 52 TNF-a Secretion by Shark Cartilage-Stimulated Leukocytes........................................ 56 IFN-y Secretion by Shark Cartilage-Stimulated Leukocytes.......................................... 62 IL-4 Secretion by Shark Cartilage-Stimulated Leukocytes............................................. 62 Apoptosis in ASC Stimulated Leukocyte Cultures.......................................................... 69 Morphological Assessment.............................................................................................69 Assessment by DNA Fragmentation Pattern................................................................ 73 Discussion.............................................................................................................................. 76 References...............................................................................................................................85 Appendices..............................................................................................................................92