Experimental Hematology Today
Experimental Hematology Today 1985 Selected Papers from the 14th Annual Meeting of the International Society for Experimental Hematology, July 14-18, 1985, Jerusalem, Israel
Edited by S. J. Baum D. H. Pluznik L. A. Rozenszajn
With 65 Illustrations
Springer-Verlag New York Berlin Heidelberg Tokyo
S. J. Baum Physiology Department Uniformed Services University of the Health Sciences Bethesda, MD 20814, USA
D. H. Pluznik Laboratory of Microbiology and Immunology National Institute of Dental Research, NIH Bethesda, MD 29782, USA
L. A. Rozenszajn Department of Life Sciences Bar-Han University, Ramat-Gan Israel
and
ISSN 0251-0170
LCCN 79-641222
© 1986 by Springer-Verlag New York Inc. Softcover reprint of the hardcover 1st edition 1986 All rights reserved. No part of this book may be translated or reproduced in any form without written permission from Springer-Verlag, 175 Fifth Avenue, New York, New York 10010, USA.
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9876543 2
ISBN-l3: 978-0-387-96273-3
DOl: 10.1007/978-1-4612-4920-7
e-ISBN-13: 978-1-4612-4920-7
Preface
Experimental Hematology Today-1985 is a memento to the superb 14th Annual Meeting of the International Society for Experimental Hematology, held in Jerusa­ lem, Israel in July 1985. It represents a selection of the best presentations at the meeting. The manuscripts were selected by the local scientific committee and care­ fully reviewed by the editors. The yearbook is divided into five parts and represents the most recent advances in the basic sciences and clinical applications.
Part I, under the leadership of Dr. L. A. Rozenszajn, is entitled "Hematopoietic Regulators." Papers in this section discuss the most recent discoveries on the phys­ iological regulation of hematopoiesis. Part II, "Hematopoietic Microenvironment," introduced by Dr. J. S. Greenberger, deals with the involvement ofthe hematopoietic microenvironment in the control of hematopoiesis. Dr. M. Saito leads Part Ill, "Dif­ ferentiation of Normal and Leukemic Cells," while Part IV, "Leukemic Cells in Leukemogenesis," is introduced by Dr. A. Raghavacher. The important discussions on recent advances in "Bone Marrow Transplantation," Part V, are headed by Dr. M. M. Bortin.
Recent findings in many disciplines in experimental and clinical hematology are presented in this yearbook. It should be of considerable value to experimental and clinical scientists.
The Editors
Part I. Hematopoietic Regulators L. A. Rozenszajn
1. Role of T-Lymphocyte Colony Enhancing Factor, TLCEF, in the Induction of CFU -TL L. A. Rozenszajn, 1. Goldman, H. Poran, M. M. Werber, D. Shoham, and 1. Radnay ........................... .
2. Thymic Hormones in Thymus Recovery from Radiation Injury R. Neta, G. N. Schwartz, T. 1. MacVittie, and S. D. Douches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3. Early Biochemical Steps in Colony Stimulating Factor (CSF) Generation are Induced by Synergy between Phorbol Esters and Calcium Ionophores D. H. Pluznik and S. E. Mergenhagen . . . . . . . . . . . . . . . . . . . 14
4. Dependence of CFU-S Proliferation on the CFU-S Population B. I. Lord. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5. Interaction of Interleukin 3 with Pluripotent Hematopoietic Stem Cells 1. L. Spivak, R. R. L. Smith, and 1. N. Ihle. . . . . . . . . . . . . . 27
6. In Vivo Effects of Urinary Extracts of Patients with Aplastic Anemia on Rat Platelet Production and Megakaryocyte Progen­ itors in Murine Spleen and Bone Marrow S-I. Kuriya, Y. Ishida, F. Ali-Osman, C. Mantel, and M. 1. Murphy lr. .................................... 33
7. Inhibitor(s) of Biologically Active Erythropoietin in Concen­ trated Human Sera 1. Barone-Varelas, C. Morley, and W. Fried . . . . . . . . . . . . . . 39
Part II. Hematopoietic Microenvironment 1. S. Greenberger
8. Establishment of Bone Marrow Stromal Cell Cultures and Per­ manent Clonal Stromal Cell Lines from Osteopetrotic (mi/mi) and Steel Mutant (Sl/Sld) Mice: Studies of Bone Resorption by
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Engrafted Hemopoietic Stem Cells In Vitro J. S. Greenberger, L. Key, C. Daugherty, J. Schwartz, and M. A. Sakakeeny .................................... 42
9. Monoclonal Antibodies Identify Specific Determinants on Re­ ticular Cells in Murine Embryonic and Adult Hemopoietic Stroma A. H. Piersma, R. E. Ploemacher, K. G. M. Brockbank, and C. P. E. Ottenheim. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
10. Stromal Cell Lines from Mouse Bone Marrow: A Model Sys­ tem for the Study of the Hemopoietic Microenvironment D. Zipori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Part III. Differentiation of Normal & Leukemic Cells M. Saito
11. Glycosphingolipids as Specific Differentiation-Markers and Differentiation-Inducers for Human Myelogenous Leukemia Cells: A Monosialyl Glycosphingolipid, Ganglioside GM3, is Highly Potent for Induction of Monocytic Differentiation of Human Myeloid and Monocytoid Cell Lines, HL-60 and U937 Cells M. Saito, H. Nojiri, and Y. Miura. . . . . . . . . . . . . . . . . . . . . . 64
12. Properties of a T-Lymphocyte Derived Differentiation Inducing Factor (OIF) for the Myeloid Leukemic Cell Line HL-60 U. Gullberg, E. Nilsson, and I. Olsson .................. 75
13. Interactions of Differentiation Inducing Agents In Vitro Provide Insight into Molecular Mechanisms of Differentiation and Iden­ tify Synergistic Combinations Effective In Vivo G. E. Francis and J. J. Berney. . . . . . . . . . . . . . . . . . . . . . . . . 82
Part IV. Leukemic Cells in Leukemogenesis A. Raghavachar
14. Immunoglobulin and T-Cell Receptor Gene Rearrangements in Human Acute Leukemias A. Raghavachar, C. R. Bartram, and B. Kubanek. . . . . . . . . . 90
15. Immunological and Molecular Classification of Human Leu­ kemias R. Foa, N. Migone, M. C. Giubellino, M. T. Fierro, P. Lusso, F. Lauria, G. Pizzolo, G. Basso, G. Cattoretti, and F. Gavosto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
16. Oncogenes in Chronic Myelogenous Leukemia R. P. Gale and E. Cannani ............................ 102
17. Response to an Active Vitamin D3 Metabolite of Transplantable Human Myeloid Leukemic Cell Lines in Adult Nude Mice G. K. Potter, A. N. Mohamed, N. C. Dracopoli, S. L. B. Groshen, R. N. Shen, and M. A. S. Moore. ... . .. 106
Part V. Bone Marrow Transplantation M. M. Bortin
18. Risk Factors for Acute Graft-vs-Host Disease in Humans M. M. Bortin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 114
19. Autologous Marrow Transplantation for Malignant Lymphoma F. R. Appelbaum, K. M. Sullivan, E. D. Thomas, C. D. Buckner, R. A. Clift, H. J. Deeg, A. Fefer, N. Flournoy, R. Hill, J. E. Sanders, P. Stewart, and
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R. Storb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 122
20. The Influence of T-Cell Depletion by Monoclonal Antibodies on Repopulating Hemopoietic Stem Cells and Their Efficacy in Preventing GvHD in Rhesus Monkeys W. R. Gerritsen, M. Jonker, G. Wagemaker, and D. W. van Bekkum..... ... ... ....... . . ... ... .... . . ... 128
21. Review of the Effects of Anti-T-Cell Monoclonal Antibodies on Major and Minor GvHR in the Mouse J/. P. OKunewick. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 133
Contributors
F. Ali-Osman, Hipple Cancer Research Center, Dayton, Ohio, and Wright State University, Dayton, Ohio, USA
Frederick R. Appelbaum, Division of Oncology, University of Washington School of Medicine, and the Fred Hutchinson Cancer Research Center, Seattle, Wash­ ington, USA
J. Barone-Varelas, Departments of Biochemistry and Medicine, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois, USA
C. R. Bartram, DRK-Blutspendezentrale, University of Ulm, D-7900 Ulm, Federal Republic of Germany
Giuseppe Basso, Dipartimento di Pediatria, University of Padova, Padova, Italy
J. J. Berney, Department of Haematology, Royal Free Hospital, London, Great Britain
Mortimer M. Bortin, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
K. G. M. Brockbank, Department of Cell Biology and Genetics, Erasmus Univer­ sity, 3000 DR Rotterdam, The Netherlands
C. Dean Buckner, Division of Oncology, University of Washington School of Med­ icine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Eli Cannani, Department of Chemical Immunology, Weizmann Institute of Science, Rehovot 76100, Israel
Giorgio Cattoretti, Laboratorio di Ematologia, I.c.P., Milano, Italy
Reginald A. Clift, Division of Oncology, University of Washington School of Med­ icine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Cathie Daugherty, Department of Radiation Oncology, University of Massachusetts Medical Center, Worcester, Massachusetts, and Joint Center for Radiation
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Therapy, Department of Radiation Therapy, Harvard Medical School, and Dana­ Farber Cancer Institute, Boston, Massachusetts, USA
H. Joachim Deeg, Division of Oncology, University of Washington School of Med­ icine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Susan D. Douches, Experimental Hematology Department, Armed Forces Radio­ biology Research Institute, Bethesda, Maryland, USA
Nicholas C. Dracopoli, Human Cancer Serology Laboratory, Sloan-Kettering Insti­ tute for Cancer Research, New York, New York, USA
Alexander Fefer, Division of Oncology, University of Washington Schooi of Med­ icine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Maria T. Fierro, Clinica Medica A, University of Torino, Torino, Italy
Nancy Flournoy, Division of Oncology, University of Washington School of Med­ icine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Robert Foa, Clinica Medica A, University of Torino, Torino, Italy
G. E. Francis, Department of Haematology, Royal Free Hospital, London, Great Britain
W. Fried, Departments of Biochemistry and Medicine, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois, USA
Robert P. Gale, Department of Medicine, UCLA School of Medicine, Los Angeles, California, USA
Felice Gavosto, Clinic a Medica A, University of Torino, Torino, Italy
W. R. Gerritsen, Radiobiological Institute TNO, Rijswijk, The Netherlands, and Primate Center TNO, Rijswijk, The Netherlands
Maria C. Giubellino, Clinica Medica A, University of Torino, Torino, Italy
J. Goldman, Department of Life Sciences, Bar-Han University, Ramat-Gan, Israel, and Department of Medical Laboratories, Meir Hospital, Kfar-Sava, Israel
Joel S. Greenberger, Department of Radiation Oncology, University of Massachu­ setts Medical Center, Worcester, Massachusetts; Joint Center for Radiation Ther­ apy, Department of Radiation Therapy, Harvard Medical School; and Dana-Farber Cancer Institute, Boston, Massachusetts, USA
Susan L. B. Groshen, Biostatistics Laboratory, Sloan-Kettering Institute for Cancer Research, New York, New York, USA
Urban Gullberg, Division of Hematology, Department of Medicine, University of Lund, 221 85 Lund, Sweden
Roger Hill, Division of Oncology, University of Washington School of Medicine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
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James N. Ihle, The National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland, USA
Y. Ishida, Hipple Cancer Research Center, Dayton, Ohio, and Wright State Uni­ versity, Dayton, Ohio, USA
M. Jonker, Primate Center TNO, Rijswijk, The Netherlands
Lyndon Key, Department of Pediatrics, Children's Hospital Medical Center, Boston, Massachusetts, USA
Bernhard Kubanek, DRK-Blutspendezentrale, University of Ulm, D-7900 Ulm, Fed­ eral Republic of Germany
S-I. Kuriya, Hipple Cancer Research Center, and Wright State University, Dayton, Ohio, USA
Francesco Lauria, Istituto di Ematologia "L. and A. Seragnoli," University of Bo­ logna, Bologna, Italy
B. I. Lord, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, Great Britain
Paolo Lusso, Clinica Medica A, University of Torino, Torino, Italy
Thomas J. MacVittie, Experimental Hematology Department, Armed Forces Radio­ biology Research Institute, Bethesda, Maryland, USA
C. Mantel, Hipple Cancer Research Center, Dayton, Ohio, and Wright State Uni­ versity, Dayton, Ohio, USA
Stephan E. Mergenhagen, Laboratory of Microbiology and Immunology, National Institute of Dental Research, NIH, Bethesda, Maryland, USA
Nicola Migone, Istituto di Genetica Medica, University of Torino, Torino, Italy
Yasusada Miura, Division of Hemopoiesis, Institute of Hematology, and Division of Hematology, Department of Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-kawachi-machi, Kawachi-gun, Tochigi-ken 329-04, Japan
Anwar N. Mohamed, Division of Neuro-Oncology, Sloan-Kettering Institute for Cancer Research, New York, New York, USA
Malcolm A. S. Moore, Laboratory of Developmental Hematopoiesis, Sloan-Ketter­ ing Institute for Cancer Research, New York, New York, USA
C. Morley, Departments of Biochemistry and Medicine, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois, USA
M. J. Murphy, Jr., Hipple Cancer Research Center, Dayton, Ohio, and Wright State University, Dayton, Ohio, USA
Ruth Neta, Experimental Hematology Department, Armed Forces Radiobiology Re­ search Institute, Bethesda, Maryland, USA
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Eva Nilsson, Division of Hematology, Department of Medicine, University of Lund, 221 85 Lund, Sweden
Hisao Nojiri, Division of Hemopoiesis, Institute of Hematology, and Division of Hematology, Department of Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-kawachi-machi, Kawachi-gun, Tochigi-ken 329-04, Japan
James P. OKunewick, Cancer Research Laboratories, Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, Pennsylvania, USA
Inge Olsson, Division of Hematology, Department of Medicine, University of Lund, 221 85 Lund, Sweden
C. P. E. Ottenheim, Department of Cell Biology and Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands
A. H. Piersma, Department of Cell Biology and Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands
Giovanni Pizzolo, Cattedra di Ematologia, University of Verona, Verona, Italy
R. E. Ploemacher, Department of Cell Biology and Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands
Dov H. Pluznik, Laboratory of Microbiology and Immunology, National Institute of Dental Research, NIH, Bethesda, Maryland, USA
H. Poran, Department of Life Sciences, Bar-Han University, Ramat-Gan, Israel, and Department of Medical Laboratories, Meir Hospital, Kfar-Sava, Israel
Gene K. Potter, Laboratory of Developmental Hematopoiesis, Sloan-Kettering In­ stitute for Cancer Research, New York, New York, USA
J. Radnay, Department of Life Sciences, Bar-Han University, Ramat-Gan, Israel, and Department of Medical Laboratories, Meir Hospital, Kfar-Sava, Israel
Anand Raghavachar, DRK-Blutspendezentrale, University of Ulm, D-7900 Ulm, Federal Republic of Germany
L. A. Rozenszajn, Department of Life Sciences, Bar-Han University, Ramat-Gan, Israel, and Department of Medical Laboratories, Meir Hospital, Kfar-Sava, Israel
Masaki Saito, Division of Hemopoiesis, Institute of Hematology, and Division of Hematology, Department of Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-kawachi-machi, Kawachi-gun, Tochigi-ken 329-04, Japan
Mary A. Sakakeeny, Department of Radiation Oncology, University of Massachu­ setts Medical Center, Worcester, Massachusetts, and Joint Center for Radiation Therapy, Department of Radiation Therapy, Harvard Medical School, and Dana­ Farber Cancer Institute, Boston, Massachusetts, USA
Jean E. Sanders, Division of Oncology, University of Washington School of Med­ icine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Gretchen N. Schwartz, Experimental Hematology Department, Armed Forces Ra­ diobiology Research Institute, Bethesda, Maryland, USA
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Joel Schwartz, Harvard School of Dental Medicine, Boston, Massachusetts, USA
Rong Nian Shen, Department of Radiation Oncology, Indiana University Hospital, Indiana University School of Medicine, Indianapolis, Indiana, USA
D. Shoham, Department of Life Sciences, Bar-Han University, Ramat-Gan, Israel, and Department of Medical Laboratories, Meir Hospital, Kfar-Sava, Israel
Robert R. L. Smith, The National Cancer Institute, Frederick Cancer Research Fa­ cility, Frederick, Maryland, USA
Jerry L. Spivak, Division of Hematology, Departments of Medicine and Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
Patricia Stewart, Division of Oncology, University of Washington School of Med­ icine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Rainer Storb, Division of Oncology, University of Washington School of Medicine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Keith M. Sullivan, Division of Oncology, University of Washington School of Med­ icine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
E. Donnall Thomas, Division of Oncology, University of Washington School of Medicine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
D. W. van Bekkum, Radiobiological Institute TNO, Rijswijk, The Netherlands, and Department of Radiobiology, Erasmus University, The Netherlands
G. Wagemaker, Radiobiological Institute TNO, Rijswijk, The Netherlands, and De­ partment of Radiobiology, Erasmus University, The Netherlands
M. M. Werber, Department of Life Sciences, Bar-Han University, Ramat-Gan, Is­ rael, and Department of Medical Laboratories, Meir Hospital, Kfar-Sava, Israel
Dov Zipori, Department of Cell Biology, The Weizmann Institute of Science, Re­ hovot 76100, Israel
I. Hematopoietic Regulators: L. A. Rozenszajn, Chairman
Role of T-Lymphocyte Colony Enhancing Factor, TLCEF, in the Induction of CFU -TL
L. A. Rozenszajn, J. Goldman, H. Poran, M. M. Werber, D. Shoham, and J. Radnay
Department of Life Sciences, Bar-Ilan University, Ramat-Gan and Department of Medical Laboratories, Meir Hospital, Kfar-Sava, Israel
ABS1RACT. T-lymphocyte colony enhancing factor lTLCEF) is a factor which is present in the con­ ditioned mediton of mononuclear cells stimulated with phytohemagglutinin (PHA). Using a prepara­ tion of partially purified TLCEF, which was devoid of other interleukin activities, it was possible to demonstrate that TLCEF was respon­ sible for the enhancement of Type I colony formation in two-step cultures. On the other hand, interleukin-2 (lL-2), and not TLCEF, was shown to be able to induce proliferation of Type II colonies even in one-step cultures, i.e., under conditions which preclude formation of Type I colonies. Individual Type I and Type II colonies were expanded in long-term culture in the presence of IL-2-containing CM. Exogenous TLCEF, unlike IL-2, was unable to support growth and recolonization of cell lines derived from individual Type I colonies. The fact that each factor seems to support the formation of a different type of colony implies that each acts either on different CFU-TL or on CFU-TL at different stages of maturation.
IN1RODUCTION
Ten years ago we developed in our laboratories cloning systems for lymphoid cells which have proved to be highly valuable for studying bio­ logical models of lymphocyte proliferation and differentiation in the irrmnme system [1]. The basic protocol for these studies was to immobil­ ize the seeded cells, usually peripheral blood mononuclear cells CMNC), bone marrow cells or lymph node cells [1-5]. The colony formation units of T-lymphocytes (CFU-TL) and B-lymphocytes (CFU-BL) were detected and monitored through their ability to proliferate in a semi-solid mediton [6,7]. In this culture system containing mitogens, with or without conditioned mediton
Send reprint requests to: Prof. L.A. Rozenszajn, Life Sciences Department, Bar-Han Uni versi ty, Ramat-Gan 52100, Israel.
1
(0.1), CFU-TL and CFU-BL progenitor cells circu­ lating in peripheral blood are able to undergo proliferation and subsequently to generate colo­ nies containing cells bearing mature T and B cell surface phenotypes, respectively.
When MNC were seeded in a two-layer agar sys­ tem, T-cell colonies developed both inside and on the surface of the upper agar layer. The lower colonies, which appeared after 3-5 days, were termed Type I, whereas the upper ones, which appeared 24-48 h later, were termed Type II.
For optimal T-cell colony growth, and in par­ ticular for Type I colonies, it was necessary to presensitize the MNC with mitogen for 18 h in liquid phase and to seed the sensitized cells in the continuous presence of mitogen [1,6]. More­ over, it was found that the addition of CM from MNC stimulated with phytohemagglutinin (PHA), enhanced the formation of colonies. The factor present in this CM which is responsible for aug­ menting the ntonber of colonies has been charac­ terized and termed T-lymphocyte colony enhancing factor (TLCEF) [8-10]. In similarity to the situation in other lineages of the hemopoietic system, we asstone that CFU-TL represent an early type of committed cell, which requires humoral regulatory factors to proliferate, differentiate and mature into T-cells. The aim of the present conununication is to shed light on the nature of the interactions between CFU-TL and TLCEF, as well as on the influence of T-cell growth factor (TCGF), also termed interleukin-2 (IL-2), on T-cell colony formation.
ME'IlIODS
COLONY FORMATION Isolation of seeded cells. The seeded cells were venous blood MNC obtained by Lymphoprep (sodium metrizoate/Ficoll, D=1.077) fractionation [11]. Two-step culture. This was performed essentially as originally described in a two-layer agar system [1], except that the teChnique was adapted to a semi -micro scale (Fig. 1). Briefly, ce11s were stimulated for 18-24 h in liquid phase with
2
MONONUCLEAR CELLS:'---I~' "'--T-MITOGEN:
101 Imi :: .'. PHA-M
1 TWO-LAYER SOFT AGAR • • • • • • • • • ••• • • • •
UPPER LAYER , ••• : ..... ': : '.: :::. " 7.5xl04 CELLS ............. .
SEMI-SOLID PHASE: T-CELL COLONY FORMATION AFTER 3-5 DAYS.
~. A schematic diagram of the semi -micro technique used for two-step culture of T­ lymphocytes.
lZ5 ug/ml PHA-M (Difco) and 10% pooled. human inactivated serum and thereafter seeded In the upper agar layer (75,000/well) in quadrupl~cates in Z4-well multidishes (Nunc), in the contInuous presence of lZ5 ug/ml PHA-M, and supplemented with ZO% pooled human inactivated serum, without or with MNC-CM or a fraction purified from it (lower agar layer). After 3-5 days at 37°C in a fully humidified atmosphere containing COz in air, Type I large colonies that had developed within the upper agar layer and had more than 50 cells were counted. Type II small and flat colonies were evaluated in some cases. One-step culture. This was performed essentially as- the two-step culture, except that the seeded cells were not stimulated with PHA in liquid phase prior to being plated in the two-layer agar system. The number of Type II colony cells was evaluated as follows: the upper agar layer on which Type II colonies had developed, was flooded with 0.5 ml of a trypsin (1:Z50) solution 0.Z5% Puck's Saline A containing EDTA (1:5000) - (Beth Haemek, Biological Industries, Israel). The tryp­ sin solution apparently caused disintegration of the colonies, and the resulting cell suspension was passed several times through a 1 ml syringe before being counted. No development of Type I colonies was observed.
PREPARATION OF MNC-CM AND A PURIFIED TLCEF FRACTION These were prepared essentially as previously reported [9,10]. Briefly, venous blood MNC (1.5x106/mI) were incubated for 72 h at 37°C in 5-7.5% COz in air in the presence of lZ5 ug/ml PHA, 5 ng/ml phorbol lZ-myristate-13- acetate (PMA) and 10% of a fraction of human serum, which was obtained as the precipitate of
fractionation (Z cycles) with 40% saturation anmonium sulfate. The CM was purified 12-Z0- fold by treating it with the ammonium sulfate solution and the supernatant, the 40S fraction containing most of TLCEF acti vi ty, was used in the experiments described in this work.
ASSAYS ~ssay. IL-Z was determined in a microassay using an IL-Z-dependent rat cytotoxic T-lympho­ cyte line [lZ]. The sample~ containing act~v~ty were tested at several dilutIonS and the actIVIty (in U/ml) was determined by logarithmically plotting the cpm of tritiated thymidine uptake against the logarithmic dilution of the sample [lZ] or by probit analysis [13]. The assay was standardized with a sample of IL-Z purified from a gibbon T-cell line, MLA-144 (a gift from Dr. H. Rabin, NCI, Frederick, MD). IL-l assay. Interleukin-l was determined using murine thymocytes as responder cells [14]. IL-3 assay. Interleukin-3 was det~rmined according to Greenberger et aL [15], USIng the murine interleukin-3-dependent line SD.
CELL LINES DERIVED FROM LYMPIDCYTE COLONIES (TYPE I AND TYPE II) Expansion and maintenance. Individual. Typ~ I al:'-d Type II colonies were expanded and maIntaIned In long-term cultures. Type I colonies were picked from the agar with a capillary tube. Type II colonies, from the surface of the agar layer, were collected by flooding the agar with RPM!- 1640 medium. The colonies were transferred, 1 colony/well, to flat-bottomed microti ter plates (Nunc), in O.Z ml complete RPMI-1640 culture medium (RPMI-1640, supplemented with 100 U/ml peniCillin, 10 ug/ml streptomycin, 1% ZOO roM glutamine, 1% 100 roM sodium '¥!:uvate, 1% non­ essential amino-acids and 5xlO- M Z-mercapto­ ethanol) containing 10% inactivated pooled human serum and ZO% MNC-CM. The cultures were incu­ bated at 37°C in a fully humidified atmosphere containing 7.5% COz in air. One half of the culture medium was replaced with fresh medium twice a week. Once a month, irradiated (3000 R) peripheral blood MNC from healthy donors were added as feeder cells at a ratio of I irradiated cell/6 cultured cells. For further maintenance, cells were transferred to Z4-well tissue dishes and the cell lines expanded under the same conditions as described above. Phenotypic analysis of cell lines. T-cell sub­ sets were determined by an indirect immunofluo­ rescence method according to their surface anti­ gen specificity using monoclonal antibodies [16].
RESULTS
In the one-step cultures, colony formation took place only when CM containing growth factors was added to the lower agar layer and essentially only Type II colonies developed [6]. In two-step cultures, the development of both Type I and Type II colonies was not entirely dependent on the addition of CM to the lower agar layer. However, the number and size of colonies was enhanced by the CM. The characteristics of the two types of
Table 1. Characteristics of Type I and Type II T- cell colonies growth in two layer soft agar culture
Colony characteristics Type I Type I I
Development in culture after 3-5 days after 5-7 days
Ce 11 content 2DO - 500 50 - 150
Morphology large, with a small, roundish compact center and flat
location in agar within the on the surface upper layer of the upper
layer
Step Purification Fraction Degree of method purification
40% ammonium 405 12 - 20 sulfate fract i onation
II Phenyl-5epharose peak II 200 - 400 chromatography
III Gel fi ltration peak 13,000 - 20,000
a. Refs 9, 10.
colonies are summarized in Table 1. Since the kinetics of appearance, the plating efficiency, the size and shape of the two types of colonies are different, it is possible that they originate from CFU-TL in different stages of maturation, and are therefore able to respond to different humoral factors.
We have attempted to identify the active sub­ stances that trigger the formation and develop­ ment of Type I and Type II colonies. The purifi­ cation of TLCEF is summarized in Table 2. TLCEF was purified up to 20,000 fold from a 3 days-CM of MNC under the synergistic stimulation of FHA and PMA [9,10]. Purified TLCEF was found to be devoid of other lymphokine activities (Table 3).
The results of one-step experiments (Table 4) show that purified IL-2, but not the fraction 405 which contains TLCEF and is IL-2 free, is able to supPort the formation of Type II colonies. The reverse is true for Type I colonies obtained in two-step cultures (Table 5): in this case TLCEF, and not IL-2, is capable of enhancing the forma­ tion of Type I colonies. It should be emphasized that under the conditions of the two-step cul­ ture, endogenous IL-2 and TLCEF are both secreted in the semi-solid medium, resulting in the forma­ tion of both Type I and Type II colonies.
Individual Type I and Type II colonies were expanded in long-term culture in the presence of IL-2-containing CM. When cell lines derived from individual Type I colonies were recolonized in agar, it was found that in the presence of fraction 405, which contains TLCEF and is free of IL-2, no colony formation took place, whereas in the presence of CM, which contains both IL-2
Table 3. Interleukin activities of partially purified TlCEF
Sample TLCEF Il_l a Il-2 1O-3x U/ml 10-3x cpm 1O-2x U/ml
CM 2.43 194.5 61.5
Phenyl- Sepharose, 0.82 3.1 0 peak IIc
Il-3b 1O-3x cpm
< 5
< 5
0
a. At a 1:8 dilution; b. At a 1:16 dilution; c. Ref 10.
Table 4. Effect of exogenous active factors on T-cell colony formation -- one step culturea
Active factor Exp't Type I Type lib No. No. of No. of
colonies colony cells
Il-2, 50 U 1 None 600,000 2 None 210,000
1l-2, 25 U 1 None 400,000 2 None 195,000
CM (containing 1 None 600,000 Il-2 and TleEF) 2 None 55,000 Fraction 40S 1 None None (containing TlCEF) 2 None 17,000
a. 3xl05 cells seeded; b. Cells of pooled colonies were scored after flooding colonies with trypsin solution.
Table 5. Enhancement effect of exogenous active factors on T-cell colony formation (Type I)
Acti ve factors
Fraction 405 (containing TlCEF)
41.0 ± 10.8
* 98.7 ± 37.3
* 90.0 ± 31.8
Results represent the mean number of colonies ± 5E of 8 separate experiments. *p < 0.05,
relative to the control.
3
and TLCEF, only flat Type II colonies neveJoped. Surface marker analysis revealed that most of the cell lines derived from Type I col.on:ies :h.ad It
heterogeneous phenotypic pattern (Fig. 2A), whereas t:hose derived from Type II colonies ,,'ere mainly eit:her OKT 4 positive or OKT 8 positive cells (Fig. 2B).
4
6 45 90 120
100
• 40 .... ~2O
CELL LINE 411
CELL LINE 5/2
DAY S OF
CELL LINE 5/1
6
~. Phenotypic analysis of 8 typical cell lines derived from the 2 types of individual T-cell colonies: A. Cell lines derived from Type I colonies; B. Cell lines deriv~d from Type II colonies. ~ OKT 8 positive cells; _ OKT 4 positive cells. At times indIcated hy arrows the cultures
were supplemented with irradiated MNC (3000 R) and PHA.
DlSQlSSION
In this work we show that in the continuous presence of a T-lymphocyte mitogen, such as PHA, CFU-TL can be induced to prol iferate in semi­ solid medium in response to stimulation by endogenous as well as exogenous growth factors present in the added CM. There seems to be no doubt that TLCEF, a factor isolated and purified from MNC-CM, is distinct from lL-2 as well as other interleukin activities (Tables 2 and 3; refs. 8-10). Other workers also have recently postulated that factors other than lL-2 may be required for in vitro proliferation, differentia­ tion and maturation of human T-colony forming cells r22-24]. The differences between nCEF and lL-2 are summarized in Table 6. The fact that each factor seems to support the formation of a different type of colony implies that each acts either on different CFU-TL or on CFU-TL in different stages of maturation [21]. Purified lL-2 is able to induce proliferation of Type II colony-forming cells, even in the one-step culture, Le., under conditions which preclude formation of Type I colonies (Table 4). On the other hand, enhancement of Type I colony forma­ tion is promoted by partially purified nCEF and not by IL-2 (Table 5). However, under all our culture conditions, in the presence of PHA, both IL-2 and TLCEF are produced endogenously. Thus, more experimental evidence is required to eluci­ date the exact nature of TLCEF action, in parti­ cular with respect to its atdlity to induce self­ renewal of the CFU-TL compartment on which it
Table 6. Comparison between characteristics of human TLCEF and IL-2
Property TLCEF IL-2
Optimal time for 48 - 72 hr 24 hra production by MNC
Additive required for None Albumin or stabi li ty at low polyethylene b protein concentration glycol (PEG)
Molecular weight 100,000-130,000 20,000-25,000 (from gel filtration)
pH stability up to 12 2 _ 10 c
Type of T -ce 11 lId colonies supported
a. Ref. 12; b. Ref. 17; c. Ref. 18; d. Refs. 19, 20, and Table 4, this work.
acts. At present, we may only speculate that TLCEF can be a differentiation and maturation factor for a population of immature IL-2- refractive T-cells. By influencing the expres­ sion of IL-2 receptors, TLCEF would render these cells responsive to the proliferative signal of endogenous or exogenous lL-2. Exogenous TLCEF, unlike lL-2, was unable to support growth and recolonization of cell lines derived from indi­ vidual Type I colonies. The lack of success in finding a population of T-cell precursors that
could he maintained in long-term culture on TLCEF alone, Le. in the ahsence of IL-2, supports the ahove hypothesis.
In conclusion, TLCEF is a factor distinct from IL-2, which seems to be required for the differentiation and maturation of premature T­ lymphocyte; it may also be needed for their pro­ liferation either alone or in combination with IL-2.
ACKIDWLEDGMENTS
We thank R. Ofir and R. Apte for performing the IL-3 assays, and L. Maron and B. Sredni for assaying IL-l. This work was supported by grants from the Israel Cancer Association and the Mitzi Dobrin Cancer Research Fund, Bar-Ilan University.
REFERENCES
1. Rozenszajn LA, Shoham D, Kalechrnan Y (1975) Clonal proliferation of PHA-stimulated human lymphocytes in soft agar culture. Immunology 29:1041
2. Riou N, Boizard G, Alcalay D, Goube de La­ forest P, Tanzer J (1976) In vitro growth of colonies from human peripheral blood lympho­ cytes stimulated by phytohemagglutinin. Ann Immunol (Inst Pasteur) l27C:83
3. Sredni B, Kalechrnan Y, Michlin H, Rozenszajn LA (1976) Development of colonies in vitro of mitogen-stimulated mouse T-lymphocytes. Nature 259:130
4. Shen J, Wilson FD, Shifrine M, Gershwin ME (1977) Select growth of human T-lymphocytes in single phase semisolid culture. J Immunol 119-1299
5. Claesson KI, Rodger MB, Johnson GR, Witting­ ham S, Metcalf D (1977) Colony formation of human T-lymphocytes in agar medium. Clin Exp Immunol 28:256
6. Rozenszajn LA, Goldman J, Kalechrnan Y, Mich­ lin H, Sredni B, Zeevi A, Shoham D (1981) T­ lymphocyte colony growth in vitro: factors modulating clonal expansion. Immunol Rev 54: 157
7. Radnay J, Goldman J, Weiss E, Rozenszajn LA (1984) Regulation of human B-cell colony growth. Cell Immunol 85:179
8. Zeevi A, Goldman J, Rozenszajn LA (1978) Partial purification and characterization of the lymphocyte colony enhancing factor (LCEF). Immunology 34:523
9. Werber t+f, Daphna D, Goldman J, Joseph D, Radnay J, Rozenszajn LA (1983) Identifica­ tion and partial purification of hurnan T­ lymphocyte colony enhancing factor (LCEF) distinct from T-cell growth factor. Immunology 50:261
10. Werber MM, Goldman J, Radnay J, Klein S, Rozenszajn LA (1985) Identification and purification of human T-lymphocyte colony­ enhancing factor, TLCEF: increased production by phorbol pyristate acetate. Immunology, in press.
11. &syurn A (1968) Separation of lymphocytes from blood and bone marrow. Scand J Clin Lab Invest 21, suppl 97:51
5
12. Stadler EM, Dougherty SE, Farrar JJ, Oppen­ heim JJ (1981) Relationship of cell cycle to recovery of IL-2 activity from human mono­ nuclear cells, human and mouse T-cell lines. J Immunol 127:1936
13. Gillis S, Ferm MM, Ou W, Smith KA (1978) T­ cell growth factor: parameters of production and a quantitative microassay for activity. J Irnmunol 120:2027
14. Gery I (1982) Production and assay of Inter­ leukin 1 (IL-l). In: Garrison F, Fitch FN (eds) Isolation, characterization and utili­ zation of T-lymphocyte clones. New York: Academic Press, p 41
15. Greenberger JS, Sakakeeny MA, Humphries PK, Eaves CJ, Bekner RJ (1983) Demonstration of a permanent factor-dependent multipotential (eosinophil/neutrophil/basophil) hematopoie­ tic progenitor cell line. Proc Natl Acad Sci USA 80:2831
16. Jannosy G, Tidman N, Papageorgiou ES, Kung PC, Goldstein G (1981) Distribution of T­ lymphocyte subsets in the human bone marrow and thymus: an analysis with mononuclear antibodies. J Immunol 126:1608
17. Mier JW, Gallo RC (1982) The purification and properties of human T cell growth factor. J Immunol 128:1122
18. Welte K, Mertelsrnann R (1985) Human Interleu­ kin 2: Biochemistry, physiology and possible pathogenetic role in immunodeficiency syn­ dromes. Cancer Invest 3:35
19. Jourdan M, Cornmes T, Klein B (1985) Control of human T-colony formation by interleukin-2. Immunology 54: 249
20. Oudnhiri N, Farcet JP, Gourdin MF, Divine M, Bouguet J, Fradelitzi D, Reyes F (1985) Mechanism of accessory cell requirement in inducing IL-2 responsiveness by human T4 lymphocytes that general colonies under PHA stimulation. J Immunol 135:1813
21. Touw I, Lowenberg B (1984) Production of T­ lymphocyte colony-forming units from pre­ cursors in human long-term bone marrow cul­ tures. Blood 64:656
22. Mossalayi MA, Goube de Laforest P, Guilhot F, Kallil G, Nytame E, Larroque V, Fellous M, Tanzer J (1985) Agar human T-cell colony growth promoted by a B+Null cell-derived lymphokine distinct from IL-2. J Inmunol 134:2400
23. Triebel F, Gluckman Je, Debre P, Charron DJ (1984) T lymphocyte progenitors in man: biochemical characterization of a colony promoting activity (CPA) active on illlllature precursors. Immunology 53:651
24. Georgoulias V, Maron S, Consolini R, Jasmin C (1985) Characterization of normal peripheral blood T- and B-cell colony-forming cells: growth factor(s) and accessory cell require­ ments for their in vitro proliferation. Cell Inmunol 90:1
Thymic Hormones in Thymus Recovery from Radiation Injury
Ruth Neta, Gretchen N. Schwartz, Thomas J. MacVittie, and Susan D. Douches
Experimental Hematology Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20814-5145, USA
ABSTRACT
The effect of a thymic hormone, thymosin fraction 5 (TF5), in restoring immuno­ competence in the thymus of y-irradiated mice was examined. Three different mouse strains were used in this study, since previous work has established that the response to TF5 varies in different strains. To measure the rate of recovery of immunocompetent cells in the thymus, the responsiveness to comitogenic effect of interleukn-l (IL-l) was used. This assay was chosen since it has been estab­ lished that only more mature PNA- , Lytl +2- medullary cells respond to this monokine. Contrary to several earlier reports that radioresistant cells repopu­ lating the thymus within the first 10 days after irradiation are mature, cortico­ steroid resistant, immunocompetent cells, the thymic cells from irradiated mice in all strains used had greatly reduced responses to IL-l. Daily intraperitoneal injections of TF5 increased significantly the responses of thymic cells to IL-l in 10-13 weeks old C57Bl/KsJ, C3H/HeJ, and DBA/l mice. Older mice, 5 months or more in age, of DBA/l strain did not respond to treatment wi th TF5. However, C3H/HeJ mice of the same age were highly respon­ sive. In conclusion, (a) cells repopu­ lating the thymus wi thin 12 days after irradiation contain lower than normal fraction of mature IL-l responsive cells, (b) thymic hormones increase the rate of recovery of immunocompetent cells in the thymus, and (c) the effect of thymic hor­ mones is strain and age dependent.
Send reprint requests to: Dr. Ruth Neta, Experimental Hematology Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20814-5145.
6
INTRODUCTION
The thymus gland is of cr i tical impor­ tance in the normal development of T-cells. T-cell precursors acquire func­ tions and phenotypic markers characteris­ tic of mature T-cells in the thymus. Much information has accumulated recently on the phenotype definition of thymic cells subpopulations [1,2] and on modes of acquisition of MHC determined self restriction necessary for their reactiv­ ity [3,4]. However, the precise intra­ thymic events that regulate thymocytes proliferation and differentiation remain unresolved. In particular, the influence of thymic hormones on proliferation and maturation of cells in the thymus remains to be established [5-7]. Much work has shown that administration of these hor­ mones in vivo can restore immunologic reactivities of immunodeficient host [8-12] • Previous work, including our own, also indicated that thymic hormones can correct deficient function but do not augment normal function [13,14]. This immunoregulatory effect awaits an expla­ nation.
Ionizing radiation, even in low doses (150-200 rad), causes a dramatic involu­ tion in murine thymus. Regeneration of the thymus, as measured by weight and mitotic index, begins 5-7 days after irradiation [15]. The thymuses of radia­ tion immunocompromised mice presented a convenient model to observe the effect of administration of thymic hormones on the matur at ion of the cells in the thymus. Two additional aspects were considered in developing the experimental model: (a) Previous work has established that the effectiveness of treatment with TF5 varies widely in different strains of mice. C3H/HeJ mice susceptible to infect­ ion with C. albicans and low responders in the in vivo release of MIF and IFN-y
become resistant and release high titers of the two lymphokines into circulation following daily administration of TF5. In contrast, DBA/l strain, also susceptib­ le and low-responder, was not affected by hormone administration [14,13]. In addi­ tion, C57Bl/KsJ, normally resistant and high responders, became susceptible and low-responders when compromised by induc­ tion of a diabetic condition [16]. This compromised strain also responded to treatment with thymosin with enhanced resistance, lymphokine release, and delayed footpad reaction to C. albicans. (b) The involution of the thymus that begins at puberty is not understood at present. It is possible that this process depends on reduced production and/or responsiveness to thymic hormones.
Therefore, our experimental model con­ sisted of the three above mentioned mouse strains, varying in their ages from 10 weeks to 6 months and irradiated with 450 rad. As a measure of thymocyte function we have chosen to assay changes in respon­ siveness to IL-l, since previous work has established that only more mature PNA-, Lytl+2- cells respond by proliferation in this assay [17-20].
In this presentation we will demonstrate that administration of TF5 into irradi­ ated mice accelerates the rate of recov­ ery of IL-l responsive cells in the thymus. The effectiveness of treatment with the hormone depends, however, on the strain and the age of the animal.
MATERIALS AND METHODS
Mice. Inbred strains of female mice (C57Bl/KsJ, DBA/IJ, and C3H/HeJ) were obtained from Jackson Laborator ies, Bar Harbor, Maine. The mice were housed in the Veterinary Medicine Department facil­ ity at the Armed Forces Radiobiology Research Institute in cages of nine mice with filter lids. Standard lab chow and HCL acidified water (pH 2.4) were given ad libi tum. All cage cleaning procedures and daily injections were carried out in a micro-isolator.
Irradiation. Mice were placed in Plexi­ glas restrainers and given whole-body irradiation at 0.40 Gy/min by bilaterally positioned cobalt-60 elements. The total dose was 4.5 Gy (450 rads).
Thymosin Fraction 5. This was obtained through the courtesy of Dr. Allan Gold­ stein, Department of Biochemistry, The George Washington University School of Medicine, Washington, DC. The control fraction, kidney fraction 5, was also kindly provided by Dr. Goldstein. Both lyophilized fractions were diluted in pyrogen-free saline (Travenol Labora­ tories) containing 100 U/ml of penicillin and 100 ~g/ml of streptomycin to a final concentration of 10 ~g/ml. Each mouse
7
received 0.5 ml daily intraperitoneal injection.
Thymic Cell Suspensions. Three to six mice per exper imental group were sacr i­ ficed via ether anesthesia on the days postirradiation as noted. Thymuses were removed, cleared of any parathymic lymph nodes and placed in Hanks Balanced Salt Solution (HBSS-GIBCO) on ice. Single cell suspensions were prepared by passing the thymuses through a Millipore screen (20 mm diameter) and then a 23 gauge needle and syringe. The cells were washed two times in HBSS (200 g, 10 min, 40C), and resuspended in complete medium con­ taining RPMI 1640, 10% calf serum (Hy­ clone), 100 u/m\ penicillin, 100 ~g/ml streptomycin, 10- M 2-beta mercaptoethan- 01, and 2 roM L-glutamine. Viability for all cell suspensions was found to always be >95%.
IL-l Preparations. Two preparations of IL-l were used. IL-l purchased from Genzyme with a specific activity of 100 U/ml was used at a final concentration of 5 U/ml and 1 U/ml (lot numbers 094a, 095a) • IL-l was also prepared in the laboratory according to Gery, et al. [21]. Briefly, resident peritoneal macrophages were lavaged from C57Bl/~ mice. Cell suspensions containing 2 x 10 cells/ml were allowed to adhere for 2 hr to the surface of plastic Costar 2506 multiwell dishes and then after removal of nonadherent cells, were incubated for 24 hr at 370C in 5% C02 with 20 ~g/ml of lipopolysaccharide (Difco, Detroit, Mich­ igan) and 60 ug/ml of silica (gift from Dr. Alison D. O'Brien, Department of Microbiology, Uniformed Services Uni­ versity of the Health Sciences) prepared as specified [22]. The supernatants were used in dilutions ranging from 1: 50 to 1:250. Controls which consisted of cell culture supernatants to which LPS and silica were added at the termination of the culture did not have any stimulatory effect.
IL-l Assays. The assay was performed as previously described [21] • Briefly, triplicate cultures for each IL-l dilu­ tion and background control were set up in 96 well flat bottom microtiter plates (Costar 3596, Cambridge, Massachusetts). Two cell concentrations were used in eac9 assay, usually 0.1 mlLwell of 3 x 10 cells/ml or 1.5 x 107 cells/mI. PHA (Wellcome Burroughs, Greenville, North Carolina) was added to the cell suspen­ sions at a final concentration of 1.0 ~g/ml. Following 48 hr incubation at 370C ~n 5% C02' cells were pulsed with 1 ~Ci H-thymidine per well. The cells were
harvested 18 hr later (Skatron Cell Harvester, Sterling, Virginia) onto glass filters which were then counted in Scintiverse II on a Mark III Scintilla­ tion Counter to determine thymidine
8
24,000
20,000
Time After Irradiation (days)
Fig. 1. Effect of TFS on comi togenic response of thymocytes from CS7Bl/6 mice. Thymocytes were recovered at days after irradiation and cultured at a cell con­ centration of 1.S x 10 6 cells/well (D + 6) or 3.0 x 10 6 cells/well (D + 9 and D + 12). Results are mean cpm of triplicate cultures with S U/ml of IL-l minus mean cpm of triplicate cultures without IL-l.
uptake. Statistical analyses were per­ formed using Student's T test.
RESULTS
. ~
10'
10'
10'
RECOVERY OF CELLS IN THE THY.MUS AFTER 4.5 Gy o·Co IRRADIATION
.-. Normal A- . -£ Thymosin
C57SLlKsJ
10·0~--~2----4L---~6L---L8--~1LO---~12----1~4--~1L6--~18 Time After Irradiation (days)
Fig. 2. The numbers of cells recovered per thymus at different days after irradiation (calculated from groups of 3-6 mice).
cell recovery in the thymus of irradiated mice [lS]. A striking difference, how­ ever, may be observed in the responsive­ ness of cells to IL-l. The TF5 treated thymocytes responded significantly more than the control and the saline treated for irradiated groups. In two additional series of experiments 10 week old mice were used, since 8-10 weeks old CD-l mice respond to IL-l with peak activity [23]. Although much higher number of cells/ thymus were recovered from the normal, 10 week old C57~1/KSJ mice (ranging from 2.3 to 4.0 x 10 cells), the thymocytes in response to 1:100 dilution of IL-l incor­ porated only 2-4 x 10 3 cprn of 3HTdR. Thus, lower levels of response were observed in thymuses with higher cellu­ larity. At day 6, 9, and 13 after irradi­ ation the TF5 treated mouse thymocytes from C57Bl/KsJ mice responded at 73%, 129 ± 7%, and 80 + 60% of normal control responses, respectively. The saline or kidney fraction S treated control groups had only 19%, 41 + 37%, and 10 + 9%, and irradiated mice had only 40%, 8 +11%, and 13 ± 8% of normal control responses on the same days. Therefore, despi te the re­ duced effect of the treatment with TF5 in 10 week old CS7Bl/KSJ mice a marked greater response was still obtained from T~S treated than from saline/kidney frac­ tlon S - treated, or irradiated mice. We conclude, therefore, that treatment with TF5 in compar ison with saline or kidney fraction S, enhances the recovery of IL-l responsive cells in the thymuses of irradiated mice.
DBA/I. Mice of this strain were evaluated since in previous experiments TF5 did not affect their resistance to infection with C. albicans and their in vivo release of IFN-y and MIF. Animals of two different ages were used to determine whether the responses to TFS are age dependent. The particular choice of ages, 10 weeks and 5 months, was based on the previous obser­ vation [23] that 8-10 week old CDFI mice
DBA/1 10 Weeks Old 100
/!.cpm 3 x 106 cells/ well O-f 9
80 cells/ thymus
.... 60 ..... 0 + 7 C 0
U :i? 0 40
0 T K R T K R
Fig. 3. Effect of TF5 on comitogenic re­ sponses of thymocytes from 10 week old DBA/l mice to IL-l. Thymocytes were recovered at 7 and 9 days after irradia­ ti~n and cultured at concentration of 3 x 10 cells/well with or without IL-l. Results are expressed as percent of con­ trol responses. T - thymosin fraction 5, k - kidney fraction 5, R - radiation only.
had maximal responses to IL-l and 18 week old mice had greatly reduced responses to purified IL-l. The lower responses in older animals may be an indication of reduced levels of immunocompetent T-cells in the thymus, possibly as a result of reduced effectiveness of thymic hormones.
(a) Thymocytes from 10 week old mice at 7 and 9 days after irradiation when treated with TF5 showed consistently higher responses (Fig. 3) • The thymocytes re­ sponses were lower in animals treated with kidney fraction 5 or irradiated only. Although lower number of cells per thymus were recovered in kidney fraction 5 and TF5 treated animals than in irradi­ ated only mice at 7 days after irradia­ tion, similar numbers of cells were re­ covered in TF5 treated group and irradia­ ted group at 9 days after irradiation. Depletion of cells, therefore, in the thymuses of TF5 treated mice does not account for the apparent difference in the level of IL-l reactive cells.
(,Q) The responses of thymocytes from 5 month old mice evaluated in two series of experiments are summarized in Fig. 4. None of the irradiated experimental groups showed the presence of IL-l re­ sponsive cells at the cell concentrations used in the assay. The cellularity of the thymuses increased with no apparent influence of treatment (Fig. 5). Thus, we can conclude that TF5, although effective in thymic recovery of younger animals, did not affect the recovery of the thymus in older animals. Cell proliferation, however, takes place in these thymuses
9
&- . _.-& Thymosin 2000 t-----t Saline -..
~ ............• '"adiation I • 1000 1
0 4 6 8 10 12 14 16 18 20
Time After Irradiation (days I
Fig. 4. Effect of TF5 on comi togenic response of thymocytes from 5 month old DBA/l mice. Thymocytes were recovered at da~s after irradiation and cultured at 3 x 10 cells/well. Results are mean cpm of triplicate cultures with 5 units of IL-l minus mean cpm of triplicate cultures wi thout IL-l.
10' . ~
E 10' >- ~
i .. 10' (,)
10' 0
RECOVERY OF CELLS IN THE THYMUS AFTER 4.5 Gy I·CO IRRADIATION
e-e Norm.' ...... Thymos;n
14 16 18
Fig. 5. The numbers of cells recovered per thymus at different days after irradiation, calculated from groups of 3-6 mice.
following radiation injury, and at 12 days the numbers of thymocytes in irradi­ ated mice nears that of normal mice.
C3H/HeJ. Mice of this strain, 5-6 months old, were used for comparison with TF5 unresponsive DBA/l mice of the same age. Two ser ies of the separate exper iments are summarized in Table 1. Since on day 6 and 7 after irradiation the recovery of cells in individual groups (6 mice in each group) was low, the ~ell concentrations were reduced to 5 x 10 cells/well on day 6 and to 7 x 105 cells/well on day 7. It is evident from Table 1 that on days 6, 7, and 9 after irradiation only thymic cells from TF5 treated mice responded to IL-l wi th increased proliferation . In con­ trast, equal cell concentrations from irradiated only, irradiated and kidney
10
EFFECT OF THYMOSIN FRACTION 5 ON COMITOGENIC RESPONSE OF THYMOCYTES FROM 5 MONTHS OLD C3HiHeJ MICE TO IL-1
TREATMENT 0+6 0+7 0+9
IL-1 CONTROL IL-1 CONTROL IL-1 CONTROL
Thymosin • 4583 ± 753 ± •• 1938 ± 432 ± t 6324 ± 1776 ±. fraction 5 2112 79 174 188 493 619
(1.0 x 1(6) (3_1 x 106) (6.5 x 107)
Kidney * 1367 ± 736 ± ... 1225 ± 540 ± t 1410 ± 1607 ± fraction 5 1000 356 203 328 95 630
(LOx 1(6) (4.1 x 106) (5.4 x 107) p <0.02 p < 0.005 p <0.01
Irradiated * 1959 ± 1713 ± .. 937 ± 540 ± t 1349 ± 1683 ± only 2050 1061 204 255 413 198
(1.0 x 106) (4.1 x 106) (5.1 x 107) p <0.05 P <0.05 P <0.001
Normal, t 4096 ± 1499± t 6162 ± 937 ± t 3252 ± 1905 ± non-irradiated 1391 801 1189 101 414 249
(7.7 x 107) (1.9 x 108) (1.5 x 108) . 685 ± 475 ± .. 264 ± 226 ± 187 112 120 77
* 5 x 105, ** 7 x 105, + 3 x 106 .
Table 1. Thymocytes were recovered at days after i~radiati~n and cultured at cell concen­ trations indicated by *(5 x 10 5), **(7 x 105 ), or T(3 x 10 ) per well. Results are me~n cpm + S.D. of triplicate cell cultures with 2 U/ml of IL-l or of controls. The numbers ln parentheses are number of cells recovered per thymus. P values were calculated by Stu­ dent's t test for a given group compared to TF5 treatment.
fraction 5 treated, and nonirradiated normal mice were not responsive to IL-l. In conclusion, unlike DBA/l mice, 5-6 month old C3H/HeJ mice respond to TF5 treatment with enhanced responses of the cells in the thymus to IL-l.
DISCUSSION
The experimental results reported here address three questions: (a) are the cells repopulating the thymus in the early postirradiation phase immunologi­ cally competent, (b) can thymic hormones exert an effect on these cells (or change their composition), and (c) can genetic factors influence the effect exerted by thymic hormones in irradiated mice.
Thymocytes from all of the strains examined, after irradiation with 450 cGy showed reduction in IL-l responsiveness throughout the first phase of recovery. Earlier studies by Takada et al. [15], using mitotic index and thymus weight from mice irradiated with 400 cGy con­ cluded that thymus regeneration is a biphasic process. Within 24 hr aftc:r irradiation, a precipitous drop ln mi tosis and in thymic we ight developed which was followed by nearly full recov­ ery beginning on days 5-7 until, day l~. Following this day a second drop ln thymlc cellularity was observed which lasted until day 20. Our observation on the recovery of cells in the thymus parallels these findings. The mechanism of this biphasic pattern of thymus repopulation remains speculative.
The immunologic competence of the cells initially repopulating the thymus is con­ troversial. A number of studies con­ cluded that radiation-resistant cells repopulating the thymus are immunocompe­ tent. Blomgren and Anderson [24] com­ pared cells from normal thymuses with corticosteroid resistant cells (CRC) for their radiation resistance. They ob­ served that corticosteroid treatment enriched the radiation resistant cells from 4% in normal mice to 50% CRC popula­ tions, and concluded that these two popu­ lations may be similar. Studies by Konda et al. [25] examining the buoyant density and T-cell markers of CRC concluded that this population was similar to radio­ resistant cells present in the thymus 10 days after 880 cGy irradiation. using 760 cGy irradiated A/J mice, Kadish and Basch demonstrated that cells recovered 9 days after irradiation had enhanced reactivity to Con-A and PHA [26]. The histology of cortical and medullary regions of the thymus 10 days after irradiation with 750 cGy resembled that of a normal thymus [27]. Therefore it has been proposed that the radioresistant cells in the thymus that repopulate the thymus after irradia­ tion present a population resembling the mature CRC. More recent studies on the phenotypes of cells present in the thymus within 12 days following irradiation demonstrated a relative sparsity of PNA-, Lytl+2- cells [28], presently recognized as the immunocompetent subpopulation that is responsive to IL-l [17-20]. Similar­ ly, CTL-precursor cells were found 14 days after lethal irradiation and bone marrow transfer but were not detected at 7 days after irradiation [29]. In another
laboratory the frequency of CTL-precursor cells was about 50-fold lower for up to 12 days after irradiation in the thymuses of bone marrow reconstituted radiation chimeras [30]. The same investigators also analyzed by flow microfluometry the Thy-l phenotype of host derived cells. Despite increasing numbers of Thy-l bright cells (considered immu~ologic~l­ ly immature), cells weakly stained with Thy-l (considered immunocompetent) were not detected 10 days after irradiation.
Our own observations using IL-l respon­ siveness as a measure of thymocyte immunocompetence indicates a reduction in these cells from day 6 to 12 postirradia­ tion with 450 cGy. Given that the number of cells recovered from the thymus at 6 days after irradiation represents about 15% of the number of cells in normal thymus and nears normal at 12 days, the frequency of these cells in the thymus must be greatly reduced. Together with the finding on reduced frequency of CTL precursor cells [29,30] and the scarcity of PNA-, Lytl+2-, weakly Thy-l stained cells repopulating the thymus after irra­ diation [28,30] the degree of maturation of radioresistant cells and the types of proliferating cells in a regenerating thymus need to be re-evaluated.
There have been numerous demonstrations that various thymic hormones preparations are effective in treatment of immuno­ deficiencies [8-14]. The capacity of these hormones to promote maturation, proliferation, and marker acquisition of T-cells in bone marrow or in splen has been reported [31-33]. However, the majority of the successful experiments demonstrating the effect of thymic hor­ mones have been conducted in vivo despite the fact that most of the in vitro experi­ ments use doses of the hormones many fold higher than the doses used in the living animal [34]. Possibly the action of this hormone is amplified in vivo via a mediat­ ing mechanism absent in the in vitro sys­ tems. The relatively narrow range of optimal doses necessary to achieve bene­ ficial in vivo effects as well as neces­ sity for daily injections of the hormone represent some of the not yet understood complexities of the system.
Although our results clearly demonstrate an enhancement of IL-l responsiveness following administration of TF5 to irra­ diated mice, the mechanism of this enhancement remains unclear. Several possibili ties should be considered. (a) Stimulation of the traffic of the bone marrow derived T-precursor cells into the thymus, (b) promotion of maturation of intrathymic cells, and (c) enrichment for IL-l reactive cells as a result of selec­ tive depletion by thymic hormones of immunologically immature cells present in the thymus. The latter possibility does
11
not seem likely as the recovery of cells per thymus on a given day does not vary much between the different exper imental groups. Treatment with TF5 enhanced the level of IL-l responsiveness in all three strains examined when the age of the mice was 10-12 weeks. The role of genetic fac­ tors is suggested by the finding that 5-6 months old C3H/HeJ mice responded to treatment (Table 1) while DBA/l mice of the same age did not respond (Fig. 4). This difference parallels the previously observed effect of TF5 in these two strains when resistance to C. albicans and in vivo release of IFN-y and MIF were compared [13,14]. The same two mouse strains also varied in their responses to IL-l. Three month old C3H/HeJ mice had tenfold higher response than 10 week old DBA/l mice (data not presented). Al­ though at 5 months the response of normal C3H/HeJ mice to IL-l had declined, it was still at least two- to threefold higher than the response of thymocytes from 5 month old DBA/l mice. This apparent dif­ ference to a comi togenic effect of IL-l may be a reflection of differences in the percentages of mature, immunocompetent cells in the thymuses of these strains. For example, the percent of medullary PNA- cells differed from 14.6% in CBA mice to 9.5% in C57Bl/6 mice [1]. Perhaps these differences in the numbers of immunocompetent cells in the thymuses of different strains may be the result of differences in the levels of endogenous thymic hormones or of cell responses to thymic hormones. The respons i veness of 5-6 month old C3H/HeJ mice to thymic hor­ mones may be the reason for this strain's high level of IL-l responses, and there­ fore the greater number of immunocompe­ tent cells in the thymus. The unrespon­ siveness of the DBA/l mice of the same age would result in lower numbers of IL-l responsive cells in the thymus of this strain as observed in the present study. As reagents to evaluate thymic hormone levels in mice of different strains become available, this hypotheses may be examined.
ACKNOWLEDGMENTS
This work was supported by the Armed Forces Radiobiology Research Institute, Defense Nuclear Agency, under Research Work Unit MJ 00148. The views presented in this paper are those of the authors: no endorsement by the Defense Nuclear Agency has been given or should be inferred. Research was conducted according to the principles enunciated in the "Guide for the Care and Use of Laboratory Animals" prepared by the Insti tute of Laboratory Animal Resources, National Research Coun­ cil. We thank Mar ianne Owens for the preparation of this manuscript.
12
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1. Sco11ay R, Shortman K (1983) Thymo­ cyte subpopu1ations: an experimen­ tal review, including flow cyto­ metric cross-correlations between the major mur ine thymocyte markers. Thymus 5:245.
2. Mathieson BJ, Fow1k1es BJ (1984) Cell surface antigen expression on thymocytes: Development and pheno­ typic expression of intrathymic subsets. Immuno1 Rev 82:141.
3. Zinkernage1 RM, Calahan GN, Klein J, Dennert G (1978) Cytotoxic T-ce11s learn specificity for self H-2 dur­ ing differentiation in the thymus. Nature 271: 251.
4. Wagner H, Hardt C, Stockinger H, pfienmainer K, Bar1et R, Ro11inghoff M (1981) The impact of the thymus on the generation of immunocompetence and diversity of antigen specific, MaC-restricted cytotoxic T-1ympho­ cyte precursors. Immuno1 Rev 58:95.
5. Low TLK, Goldstein AL (1982) Role of the thymosins as immunomodu1ating agents and maturation factors. In Maturation Factors and Cancer, Moore MAS, ed. Raven Press, New York p. 229.
6. Ho AD, Ma DDF, Price G, Hunstein W, Hoffrand AV (1983) Biochemical and immunological differentiation of human thymocytes induced by thymic hormones. Immuno1 50:471.
7. Andrews P, Shortman K, Sco11ay R, Potworowski EF, Kruisbeek AM, Go1d­ ste in G, Trainin N, Bach JF (1985) Thymus hormones do not induce pro­ liferative ability or cytolytic function in PNA+ cortical thymo­ cytes. Cell Immuno1 91:455.
8. Wara DW, Goldstein AL, Doyle N, Ammann AJ (1975) Thymosin activity in patients with cellular immuno­ deficiency. New Eng J Med 292:70.
9. Morrison NE, Collins FM (1976) Restoration of T-cell responsiveness by thymosin: Development of anti­ tuberculous resistance in BCG in­ fected animals. Infec Immun 13:554.
10. Mawhinney H, G1eadhi11 VF, McCrea S (1979) In vitro and in vivo re­ sponses to thymosin in severe com­ bined immunodeficiency. C1in Immuno1 Immunopatho1 14:196.
11. Bonagura VR, Pitt J (1981) Hypoparat­ hyroidism with T-cell deficiency and hypoimmunog1obu1inemia: Response to thymosin therapy. C1in Immuno1 Immunopatho1 18:375.
12. Petro TM, Chien G, watson RR (1982) Alteration of cell mediated immunity to Listeria monocytogenes in protein ma1nurished mice treated with thymos­ in fraction 5. Infec Immun 35:601.
13. Neta R, Salvin SB (1983) Resistance and susceptibility to infection in inbred murine strains. II. Var ia­ tions in the effect of treatment with thymosin fraction 5 on the
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release of 1ymphokines in vivo. Cell Immuno1 75:173. Salvin SB, Neta R (1983) Resistance and susceptibility to infection in inbred murine strains. I. Variat­ ions in the response to thymic hor­ mones in mice infected with Candida a1bicans. Cell Immuno1 75: 160. Takada A, Takada Y, Huang CC, Ambrus JL (1969) Biphasic pattern of thymus regeneration after whole body irra­ diation. J Exp Med 129:445. Salvin SB, Neta R (1984) The in vivo effect of thymosin on cell-mediated immuni ty. In Thymic Hormones and Lymphokines, Goldstein AL, ed. Plenum, New York, p. 439. Oppenheim JJ, Northoff H, Greenhill A, Mathieson BJ, Smith K, Gillis S (1980) Properties of human monocyte derived lymphocyte activating factor (LAF) and lymphocyte derived mito­ genic factor (LMF). In Biochemical Characterization of 1ymphokines, DeWeck AL, Kristensen F, Landy M, eds, Academic Press, New York, p. 399. Oppenheim JJ, Stadler BM, Siraganian RP, Mage M, Mathieson BJ (1982) Lymphokines: Their role in lympho­ cyte responses. Properties of inter1eukin-1. Fed Proc 41:257. Conlon PJ, Henney CS, Gillis S (1982) Cytokine-dependent thymocyte responses: Characterization of IL-1 and IL-2 target subpopu1ations and mechanism of action. J Immuno1 128:797. Puri J, Shinitzky M, Lonai P (1980) Concomitant increase in antigen binding and in T-cell membrane lipid viscosity induced by the lymphocyte acti vating factor, LAF. J Immuno1 124:1937. Gery I, Davies P, Derr J, Krett N, Barranger JA (1981) Relation between production and release of lymphocyte act~vating factor (inter1eukin-1) by mur l.ne macrophages. I. Effects of various agents. Cell Immuno1 64: 293. O'Brien AO, Scher I, Formal SB (1975) Effect of silica on the innate resistance of inbred mice to Salmonella typhimurium infection. Infec Immun 25:513. B1yden G, Handschumacher RE (1977) Purification and properties of human lymphocyte activating factor (LAF). J Immuno1 118:1631. Blomgren H, Andersson B (1970) Characteristics of the immunocompe­ tent cells in the mouse thymus: Cell population changes during cortisone­ induced atrophy and subsequent regeneration. Cell Immuno1 1:545. Konda S, Stockert E, Smith RT (1973) Immunologic properties of mouse thymus cells: Membrane antigen patterns associated with various
cell subpopulations. Cell Immunol 7:275.
26. Kadish JL, Basch RS (1975) Thymic regeneration after lethal irradia­ tion: Evidence for an intrathymic radioresistant T-cell precursor. J Immunol 114:452.
27. Sharp JG, Thomas DB (1975) Thymic regeneration in lethally x-irradi­ ated mice. Radiat Res 64:293.
28. Sharrow SO, Singer A, Hammerling U, Mathieson BJ (1983) Phenotypic characterization of early events of thymus repopulation in radiation bone marrow chimeras. Transplanta­ tion 35:355.
29. Korngold R, Bennink JR, Doherty PC (1981) Early dominance of irradiated host cells in the responder profiles of thymocytes from P-Fl radiation chimeras. J Immunol 127:124.
30. Ceredig R, MacDonald HR (1982) Phenotypic and functional properties of murine thymocytes. II. Quantitat­ ion of host- and donor-derived cyto­ lytic T-lymphocyte precursors in regenerating radiation bone marrow chimeras. J Immunol 128:614.
31. Bach JF, Dardenne M, Goldstein AL, Guha A, White A (1971) Appearance of T-cell markers in bone marrow rosette-forming cells after incuba­ tion with thymosin, a thymic hor­ mone. Proc Natl Acad Sci USA 68: 2734.
32. Pazmino NH, Ihle IN, Goldstein AL (1978) Induction in vivo and in vitro of terminal dioxynucleotidyl transferase by thymosin in bone marr­ ow cells from athymic mice. J Exp Med 147:708.
33. Goldschneider I, Ahmed A, Bollum FJ, Goldstein AL (1981) Induction of terminal deoxynucleotdyl transferase and Lyt antigens with thymosin. Identification of multiple subsets of prothymocytes in mouse bone mar­ row and spleen. Proc Natl Acad Sci USA 78:2469.
34. Zatz MM, Oliver J, Samuels C, Skot­ nicki AB, Sztein MB, Goldstein AL (1984) Thymosin increases production of T-cell growth factor by normal human peripheral blood lymphocytes. Proc Natl Acad Sci USA 81: 2882.
l3
Early Biochemical Steps in Colony Stimulating Factor (CSF) Generation are Induced by Synergy between Phorbol Esters and Calcium Ionophores
Dov H. Pluznik and Stephan E. Mergenhagen
Laboratory of Microbiology and Immunology, National Institute of Dental Research, NIH, Bethesda, Maryland 20892, USA
ABSTRACT. In many secretory systems receptor triggering by agonists is followed by inositol phospholipid breakdown to diacylglycerol (DAG) and inositol triphosphate (InsP3). DAG activates protein kinase C (PK-C) and InsP3 mobilizes intracellular calcium. Both tumor promoting phorbol esters (TPA) which activate PK-C directly and calcium ionophores which mobilize intracellular calcium bypass inositol phospholipid breakdown. We recently reported that the interaction of TPA and bacterial lipopoly­ saccharide (LPS) with murine bone marrow cells (BM) is followed by generation of CSF. Optimal generation occurs when TPA and LPS are added simultaneou~ly. To determine whether generation of CSF requires activation of PK-C and calcium mobilization we tested the ability of A23187, a calcium ionophore, to replace LPS. BM and spleen cells were stimulated with TPA and A23187 and the supernatants were assayed for CSF by measuring 3H-thy­ midine incorporation into a CSF dependent basophil/mast cell line, PT-18. TPA and A23187 acted cooperatively to stimulate generation of CSF similar to the action of TPA and LPS. In addition, trimethoxyben­ zoate (TMB-8), an inhibitor of calcium mobilization, inhibited CSF production either by TPA and LPS or by TPA and A23187. Synthetic DAG was able to replace TPA in stimulating spleen cells together with A23187 to generate CSF. Generation of CSF by spleen cells can be inhibited by TMB-8 only when added to the cells up to 10 min after stimulation with TPA and A23187. Later addition of TMB-8 had no effect. The results reported suggest that calcium mobilization and activation of PK-C are early biochemical events in the sequence leading to the generation of CSF.
Key words: phorbol esters - inositol triphosphate - CSF - protein kinase C - calcium mobilization
14
Antigens and lectins can stimulate lymphoid cells from peripheral organs to produce colony stimulating factors (CSF). Bone marrow cells (BM), which contain the target cells for CSF, do not produce CSF in response to such stimuli. We have recently reported that the synergistic interaction of bacterial lipopolysac­ charides (LPS) and tumor promoting phorbol esters (TPA) with murine BM is followed by the generation of CSF (1,2). Recent studies have suggested that the inter­ action of a wide variety of biologically active substances with their specific cell surface receptors is followed by an immediate breakdown of membrane inositol phospholipid which is associated with an increase in intracellular calcium (3,4). These biochemical events seem to mediate many physiological responses of cells. Two of the main products of the breakdown of inositol phospholipids are the transiently produced diacylglycerol (DAG) and inositol triphosphate (InsP3). DAG operates within the plane of the membrane and activates the calcium dependent enzyme, protein kinase C (PK-C), whereas InsP3 is released into the cytoplasm to function as a second messenger for mobilizing intracellular calcium. PK-C is now widely accepted to be the cellular target for TPA, which bypasses the inositol phospholipid breakdown and interacts directly with the enzyme (Fig. 1) •
In view of these observations, we postulated that the cooperation between LPS and TPA in stimulating BM cells to generate CSF may be linked to activation of PK-C by TPA and to calcium mobilization by LPS. Furthermore, we questioned whether the early steps in the generation of CSF require the activation of PK-C and calcium mobilization. To elucidate such a possi­ bility we tested the ability of the calcium ionophore, A23187, which can abolish the effects of InsP3 by discharg­ ing intracellular calcium stores from
PhorbO~resters-,-l-__________ _
Exogenous OAG
/ Inositol
breakdown
Physiological responses (CSF)?
endoplasmamic reticulum (5), and of TPA which can directly activate PK-C (6), to stimulate BM and spleen cells to produce CSF and thus bypass the requirement for antigen or lectin for such a stimulation.
MATERIALS and METHODS
Mice: CBA/J male mice 8-16 weeks old were used in all experiments (Jackson Laboratory, Bar Harbor, ME).
Chemicals: 12-0-tetradecanoyl-phorbol-13- acetate (TPA) (Consolidated Midland Co., Brewster, N.Y.), was dissolved in dimethyl sulfoxide (DMSO) to 2mM and stored at -70·C. Calcium ionophore A23187 (Calbio­ chem, La Jolla. CA) was dissolved in DMSO to 1mM and stored at -70·C. 3,4.5-tri­ methoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8) (Sigma Chemical Co .• St. Louis. MO) was dissolved in DMSO to 100mM and stored at -20·C. Synthetic 1-oleoyl-2 acetyl glycerol (DAG) (Molecular Probes, Inc .• Junction City. OR) was dissolved in DMSO to 25 ~g/ml and stored at -20·C. The final concentration of DMSO in the cultures in any combination of chemicals was always less than 1% and usually between 0.1% and 0.05%. LPS from salmonella abortus equi-W (Difco, Detroit MI) was reconstituted with distilled water (1 mg/ml) and stored at -20·C. Before addition to cells all chemicals were diluted in growth medium.
Growth media: Two growth media were used; a) Dulbecco's modified Eagle's medium (DMEM) supplemented with heat inactivated horse serum (GIBCO, Grand Island. N.Y.) was used for BM cells. b) RPMI-1640 medium supplemented with 1mM L-glutamine. 1mM pyruvate. nonessential amino acids at O.lmM each. 25mM hepes buffer, 50~M mercaptoethanol and 10% fetal calf serum was used for spleen cells and for the T cell lines.
Cell lines: PT-18 basophil/mast cell line (7) was maintained by twice per week passage of the cells in RPMI-1640 growth medium to which 20% of spleen concanavalin A (Con A) conditioned medium was added.
15
Fig. 1. The proposed role of phorbol esters and exogenous DAG in activa­ tion of protein kinase C and of A23187 as a calcium mobilization agent. Similar proposed' roles are attributed to the intracellular physiologic breakdown products of inositol phospholipid. DAG and inositol triphosphate (Insp3).
The EL-4 thymoma cell line (8) and the 2C2.45.5 hybridoma T cell line (9) were maintained by twice per week passage of the cells in RPMI-1640 growth medium.
Preparation of CSF: CSF was prepared from BM. spleen, EL-4 and 2C2.45.5 cells. The cells were stimulated with different combinations of TPA. LPS and A23187 as indicated in each experiment. These chemicals were added to the cells for 4 hours at 37·C, after which the cells were washed with DMEM (BM cells) or RPMI-1640 medium (spleen and T cell lines) and resuspended in fresh growth medium for an additional 20 hours at 37·C. After incubation. the cells were centrifuged and the supernatants were assayed for CSF.
Assay of CSF activity: Proliferation of cells of a GM-CSF-dependent cell line, PT-18, was used to quantitate CSF activity (7,10). Briefly, supernatants contai~ing CSF were added to PT-18 cells (5 x 10 /0.2 ml per well) for 40 hours. 3The cells were pulse labelled with 1 ~Ci H-thymidine (1.9 Ci/mol, Amersham) as a measure of cellular DNA synthesis. CSF activity was expressed as stimulation index which is the ratio between cpm obtained in cells stimulated with CSF to cpm obtained in cells incubated in medium alone.
RESULTS and DISCUSSION
BM cells stimulated with optimal concen­ trations of TPA (10-6 M) and LPS (12.5~g/ml) produced CSF. However, when each of the chemicals was added consecutively only small amounts of CSF were generated. Addition of either chemical alone did not stimulate CSF production (Fig. 2). The synergistic effect of the combination of TPA and LPS to stimulate BM cells to produce CSF resembled the synergistic effect of activation of PK-C activation and calcium mobilization in platelets and neutrophils necessary to release serotonin (11) and lysosomal enzymes (12). Since TPA is known to activate PK-C, we reasoned that LPS may be mobilizing calcium in BM cells. In the next group of experiments, we
16
TPA + LPS, 2hr
TPA + LPS, 4hr
STIMULATION INDEX
150
Fig. 2. Synergistic effect of TPA (l0-6M) and LPS (12.5 \lg/ml) in stimulating BM cells (5xl0 6 /ml) to produce CSF.
125
j OJ
Ca'o- IONOPHORE 1M)
Fig. 3. Synergistic effect of TPA (10-6M) and calcium ionophore (A23187) in stimulating BM cells (5xl0 6 /ml) to produce CSF.
replaced LPS with the calcium ionophore A23l87. Together with TPA, A23187 (Fig. 3) stimulated BM cells to produce CSF' 7 The A23187 at concentrations of 5 x 10- M to 5 x 10-6 M stimulated BM cells in the presence of 10-6 M TPA to produce CSF. A23187 alone stimulated the production of only small amounts of CSF. Under the same experimental conditions when BM cells were replaced by spleen cells the combination of TPA and A23187 also was effective in inducing spleen cells to generate CSF (Fig. 4). However, while TPA or A23187 alone stimulated only very small amounts of CSF in BM cultures, each of the two chemicals independently stimulated the production of relatively high amounts of CSF from spleen cells. However, a much higher yield of CSF was produced when a combination of both chemicals was used
300 C"'-lonophoreIM)
1.25)( 10-7 2.5 X 10-' 5 )( 10-7 10"' TPA 1M)
Fig. 4. Synergistic effect of TPA and calcium ionophore (A23l87) in stimulating spleen cells (5xl0 6 /ml) to produce CSF.
with the spleen cells. This difference between the spleen and BM could be due to the fact that the spleen contains mature cells which can replace to a certain degree the activity of either TPA or A23187. Moreover, some cellular processes, such as secretion in blood platelets can be activated through PK-C pathway without a change in resting levels of calcium (13), but a more effective stimulus is provided when both pathways (PK-C and InsP3) act in concert.
BM and spleen contain a heterogeneous cell population. It is difficult, there­ fore, to demonstrate directly calcium mobilization by measuring quin-2 fluores­ cence (14). In the next series of experi­ ments we used TMB-8, an inhibitor of calcium mobilization (15), to test whether calcium mobilization is an essential step in the triggering of cells to produce CSF. BM and spleen were pre-incubated for 30 minutes with increasing concentrations of TMB-8 before the addition of TPA (10- 6 M) and LPS ;12.5jJg/ml) to BM cliis or TPA (2 x 10- M) and A23l87 (10~ M) to spleen cells. Fig. 5 (for BM cells) and Fig. 6 (for spleen cells) show the results of such experiments. TMB-8 at 300jJM reduced CSF production significantly by both BM and spleen cells. At lower concentrations of TMB-8 only a partial reduction of CSF production was observed. Recently, it was reported that TMB-8 in addition to inhi­ biting calcium mobilization can also inhibit PK-C activity (16). To test whether the inhibitory action of TBM-8 on BM and spleen cells is mainly on calcium mobility or also on PK-C activation, the following experiments were undertaken. A T cell line, a thymoma (EL-4), was stimulated with the PK-C activator TPA (2x 10-8 M) and a T cell hybridoma (2C2.45.5), was stimulated with the calcium ionophore A23187 (10-7 M). Both cell lines produced CSF in amounts similar to those produced by spleen cells
100
75
100 300 500 700 TMB-8I~M)
Fig. S. Dose dependent inhibition of TMB-8 on CSF production by BM (Sx10 6 /ml) synergistically stimulated with TPA (10- 6M) and LPS (17.5 ~g/ml).
stimulated with TPA and A23187 (data not shown). TMB-8 at various concentrations was added to these two T cell lines and the inhibitory effect on CSF production was evaluated. From these results the amounts of TMB-8 inhibiting 50% (rOSO) of CSF activity were calculated and compared to the roso values affecting the product­ ion of CSF by BM cells stimulated with TPA and LPS and by spleen cells stimulated with TPA and A23187. The roso concentrat­ ions of TMB-8 which inhibited CSF product­ ion by BM cells stimulated with TPA and LPS and by spleen cells stimulated with TPA and A23187 were similar to the r050 inhibiting CSF production by the hybridoma cell line (2C2.4S.S) stimulated w~th A23187 alone (Fig. 7). These concen­ trations were about 100~M TMB-8, while the roso inhibiting CSF production by the thymoma cell line (EL-4) stimulated by TPA alone was about 3S0~M. This significant difference between the roSo required to inhibit CSF production when stimulated by TPA to the r050 required to inhibit CSF production when stimulated by A23187 suggests that the main target of the TMB-8 inhibition in BM and spleen cells is the calcium mobilization. However, based on the results shown in Fig. 6, the inhibit­ ion of activation of PK-C cannot be entirely ruled out.
1,2 diacyglycerol is produced from hydrolysis of inositol phospholipid and serves as the endogenous activator of PK-C (3). Thus, in the next group of experi­ ments we tested whether TPA can be re­ placed by synthetic OAG (100~g/ml) in stimulating spleen cells together with A23187 to generate CSF. OAG could replace TPA in stimulating spleen cells to produce
z a E CD I ;;;;
100
100 300 500 700 TMB-81~M)
Fig. 6. Dose dependent inhibition of TMB-8 on CSF production by spleen cells (Sx10 6 /ml) synergistically stimulated with TPA (2x10- 7M) and A23187 (10- 6M).
17
CSF (Fig. 8). rt was most effective with the lower 6 concentrations of A23187 (5x10~ M and 10 - M) tested. OAG at a concentra­ tion of 100~g/ml was optimal in stimula­ ting spleen cells to generate CSF; higher or lower doses were less effective (data not shown). OAG was less effective than TPA in stimulating spleen cells to generate CSF. This could be due to the difficulty in dispersing it in a form suitable for presentation to the cells (3). rn addition TPA is much more potent than OAG in activating PK-C, probably because it is not inactivated by the OAG kinase of the stimulated cells (11).
Finally, TMB-8 was used to determine the length of time by which spleen cells have to be stimulated by TPA and A23187 to produce CSF. This could not be effective­ ly achieved by washing the spleen cells from TPA and A23187 since the possibility exists that the chemicals are bound to the cells and cannot be removed completely by washing. TMB-8 provided us with an experimental tool to measure the time necessary for stimulation. Spleen cells were stimultted with TPA (2 x 10- 7 M) and A23187 (10- M) and at various time inter­ vals TMB-8 at a concentration of 300~M was added. After incubation of the cells for a total of 4 hr, the cells were washed and further incubated for an additional 20 hr in growth medium. When TMB-8 was added to spleen cells 10 min after the addition of TPA and A23187, almost no inhibition of production of CSF was observed (Fig. 9). TMB-8 was effective in reducing CSF production only when added during the first 10 minutes. Thus, mobilization of calcium can be considered as an early step in triggering the cells to generate CSF.
18
Thymoma TPA IEL4)
50
Ca~~ IONOPHORE (M)
Fig. S. CSF production by spleen cells (5xI0 6 /ml) after stimulation with syn­ thetic l-oleoyl-2-acetyl glycerol (DAG. 100~g/ml) and the calcium ionophore A231S7.
300
The synergistic role of PK-C and calcium mobilization has been shown in several systems such as in the release of serotonin from platelets (II). release of histamine from mast cells (17). release of lysosomal enzymes from neutrophils (12). release of catecholamine from bovine adrenal medullary cells (IS). secretion of aldosterone from porcine adrenal glomerulosa cells (19) and release of insulin from rat pancreatic islets (20) and in the present study. the release of CSF from BM cells and spleen cells. The results reported here also show that calcium ionophore in conjunction with TPA can mimic the effect of antigens and lectins in stimulating spleen cells to produce CSF. These results suggest. therefore. that antigenic and mitogenic activation of