University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 1998 Analysis of eicosanoids released from activated macrophages Rong Wang The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Wang, Rong, "Analysis of eicosanoids released from activated macrophages" (1998). Graduate Student Theses, Dissertations, & Professional Papers. 6653. https://scholarworks.umt.edu/etd/6653 This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. I s,TO Maureen and Mike MANSFIELD LIBRARY The University of IMONXANA Pennission is granted by the author to reproduce this material in its entirety, provided that this material is used for scholai'ly purposes and is properly cited in published works and reports. PlcûSc check "Yes'' or "No" and provide signature«* Yes, I grant permission No, I do not grant permission Author’s Signature Date Any copying for commercial purposes or financial gain may be undertaken only with the author's explicit consent. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ANALYSIS OF EICOSANOXDS RELEASED FROM ACTIVATED MACROPHAGES By Rong Wang B.S., Shanghai Second Medical University, 1992 Presented in partial fulfillent of the requirements for the degree of Master of Science University of Montana 1998 Approved by ard of Examiners \J L i) Dean, Graduate School Date Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: EP37454 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMT Di*Mrtation PiAJiahing UMI EP37454 Published by ProQuest LLC (2013). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 -1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Wang, Rong., M.S., May 1998 Microbiology Analysis of eicosanoids released from activated macrophages (94 pp.) Director: George L. Card Macrophages are a major source of the eicosanoid derivatives of arachidonlc acid, which mediate a number of reactions that have important biological functions. In this study, we developed a C18 minicolumn procedure for rapidly separating eicosanoid mixtures into prostaglandin, leukotriene, and arachidonic acid fractions. The procedure was then used to quantify eicosanoids released from macrophages (both human THP-1 cells and mouse peritoneal exudate cells) activated with different bacterial amphiphiles (lipopolysaccharide (LPS), lipoteichoic acid (LTA), and monophosphoryl lipid A), ceramide, and phorbol myristate acetate (PMA). Arachidonic acid was the main product in eicosanoid mixtures released from both cell lines. The prostaglandin pathway was activated in mouse PEC, but not in human THP-1 cells. There were marked differences in the relatgive potency of different amphiphile preparations for the activation of both cell types. Pretreatment of THP-1 cells with 7-interferon was not required for the activation of arachidonic acid metabolism in the human THP-1 cell line. Treatment with R595 LPS desensitized mouse PEC to subsequent challenge with R595 LPS, but not with either C2-ceramide or PM A. HPLC analysis confirmed that arachidonic acid was the main product released from activated THP-1 cells and suggested that the eicosanoid mixtures released from acitivated cells contained products other than prostaglandins, leukotrienes, and arachidonic acid. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS With my sincerest gratitude and appreciation, I would like to thank my advisor. Dr. George Card, for his guidance, patience, and friendship throughout my studies at the University of Montana. His advice, support and encouragement made the completion of this study possible. He has also seen me through many difficult times in my life and has helped me grow as a student and a person. Also, I would like to thank the members of my Master’s committee. Dr. Michael Minnick and Charles Eyer for their invaluable advise and suggestions. Thanks to the Stella Duncan Research Institute and Ribi ImmunoChem Research Inc. for their support. I also wish to give special thanks to Jean Pfau for her continued patience and help that she has provided to me during the time I worked with her in Dr. Card’s laboratory. Finally, but most importantly, I dedicate this work to my family - my parents and grandmother. Their love, encouragement, and silent sacrifices over the years have helped me through thick and thin, and gave me strength throughout the study. With love I would like to thank my dear grandmother, who passed away during the time when I was working on my degree. Her unlimited love, support, understanding and guidance have made me who I am today. I love her dearly. I l l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. t a b l e o f c o n t e n t s Chapter I. Introduction Arachidonic a c i d .......................................... 3 Leukotrienes .............................................. 4 Prostaglandins ............................................ 6 Bacterial amphiphiles .................................... 11 1) lipopolysaccharide ................................... 11 2) Monophosphoryl lipid A ............................... 13 3) lipoteichoic a c i d .................................... 14 Objectives ............................................... 17 Specific aims ............................................ 17 Chapter II. Materials and Methods Materials ................................................ 19 Fractionation of eicosanoids on CIS minicolumns .......... 19 Macrophage cultures ...................................... 21 1) Mouse peritoneal exudate cells ....................... 21 2) Human THP-1 cells .................................... 21 Bacterial amphiphiles .................................... 22 Labeling procedure ....................................... 22 Macrophage challenge ..................................... 23 HPLC analysis ............................................ 24 Chapter III. Results Binding affinity of different eicosanoids on CIS minicolumns ...................................... 25 Binding affinity of different eicosanoids on silicic acid columns .................................. 2 6 Separation of leukotrienes, prostaglandins, and arachidonic acid on CIS minicolumns .................. 27 Separation of leukotrienes, prostaglandins, and arachidonic acid on silicic acid columns ................ 30 The relative amount of label in the different eicosanoid fractions released from LPS activated THP-1 cells was similar throughout the activation period ........................................ 35 IV Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. THP-1 macrophages activated with LPS from S. minnesota (R595) released both PGE2 and LTC4 ....................... 3 9 The kinetics of activation of THP-1 cells with lipoteichoic acid were similar to the kinetics of cells activated with LPS .............................. 42 The relative amount of label recovered in different eicosanoid fractions was similar for THP-1 cells activated with different LTA preparations ............... 45 Pretreatment of THP-1 cells with human Gamma Interferon did not alter the pattern of arachidonic acid metabolism............ 48 Mouse peritoneal macrophages (PEC) were more sensitive to PMA than human THP-1 macrophages ...................... 50 Comparison of the effects of C2-ceramide, sphingomyelinase, and dihydroceramide on arachidonic acid metabolism in mouse peritoneal macrophages (PEC).............................. 55 Effect of R595 LPS pretreatment of macrophage cultures (Mouse PEC) on subsequent challenge with S.minnesota LPS, ceramide, and phorbol myristic acetate (PMA) ............ 57 HPLC analysis ............................................ 61 Membrane phospholipid was not recovered in eicosanoid fractions released from activated human THP-1 cells ..... 64 Chapter IV. Discussion Development for rapid separation of different eicosanoids .............................................. 68 Comparison of eicosanoid separation efficiency of silicic acid column procedure and CIS minicolumn procedure ................................................ 69 LPS and LTA activated eicosanoid metabolism in human THP-1 cell line .................................... 70 S.minnesota LPS, S .faecalis LTA, and PMA activated eicosanoid metabolism in mouse