Experimental Hematology Today—1985: Selected Papers from the 14th Annual Meeting of the International Society for Experimental Hematology, July 14–18, 1985, Jerusalem,
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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.
The use of general descriptive names, trade names, trademarks,
etc., in this publication, even if the former are not especially
identified, is not to be taken as a sign that such names, as
understood by the Trade Marks and Merchandise Marks Act, may
accordingly be used freely by anyone.
While the advice and information of this book is believed to be
true and accurate at the date of going to press, neither the
authors nor the editors nor the publisher can accept any legal
responsibility for any errors or omissions that may be made. The
publisher makes no war ranty, express or implied, with respect to
material contained herein.
Printed and bound by Edwards Brothers, Inc., Ann Arbor, Michigan.
Printed in the United States of America.
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
vii
viii
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
ix
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
xi
xii
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
xiii
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
XIV
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
xv
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
REFERENCES
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
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
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
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