liver, lungs, and peritoneal cavity. (Pe). The phagocytosis of sheep red blood cells. (SRBC) by these. M was normal or even en- hanced, as in the case of Pe-M.
Journal
of Leukocyte
Biology
46:134-143
(1989)
Macrophage Function in Murine Allogeneic Bone Marrow Radiation Chimeras in the Early Phase After Transplantation Joachim
Roesler, Department
Manuela
Baccarini,
Birgit
Vogt,
and
of Immunbiology, Fraunhofer-Institute for Toxicology Hannover, Federal Republic of Germany
Marie-Luise and
Aerosol
Lohmann-Matthes Research,
We tested several of the functions of macrophages (M’) in the early phase after allegeneic bone marrow transfer to get information about this important aspect of the nonspecific immune system in the T-cell-deficient recipient. On days 3-5 after transfer, the number of M4 was reduced in the spleen, liver, lungs, and peritoneal cavity (Pe). The phagocytosis of sheep red blood cells (SRBC) by these M was normal or even enhanced, as in the case of Pe-M. Already on days 8-12 after transfer, the number of Md, in spleen and liver exceeded that of controls, whereas the number was still reduced in lungs and Pe. We examined their ability to kill P815 tumor cells, to produce tumor necrosis factor-u (TNFu), to phagocytose SRBC, to produce reactive oxygen intermediates (AOl) in vitro and to kill Listeria monocytogenes in vivo. Most functions were normal and often even enhanced, depending on the organ origin, but the ability of Pe-M4 to produce AOl was reduced. Proliferative response to macrophage colony-stimulating factor (MCSF) and killing of YAC-1 tumor cells revealed a high frequency of macrophage precursor cells in the spleen and liver and a high natural killer (NK) activity in the liver. Altogether, enhanced nonspecific immune function, especially preactivated Md,, may enable chimeras to survive attacks by opportunistic pathogens. Key words:
aliogeneic bone marrow transfer, phagocytosis, ygen intermediates, Listens monocytogenes
INTRODUCTION It is well known that immediately after clinical or experimental bone marrow transplantation the immune system fails to mount any antigen-specific immune response, due to a lack of functional T cells. Recovery from this serious defect in defense capability proceeds slowly in the case of fully allogeneic transplantation and may even remain finally incomplete [e.g., 8, 16]. The fate of treated individuals ultimately depends on socalled nonspecific immune functions, which have not yet been studied in detail in the period immediately following transplantation. We report that fully allogeneic radiation chimeras can survive under conventional conditions without antibiotics (survival rate about 70%) if graft-vs. -host disease (GVHD) is prevented. They demonstrate good protection against endemic opportunistic pathogens despite the fact that a complete lack of T-cell function can be shown clearly in the combination of mouse strains used (e.g., lack of alloreactive T cells, no antibody production against SRBC in the early phase). Our studies reveal that not only specific but also certain nonspecific immunologic functions are impaired in © 1989
Alan
A. Liss,
Inc.
tumor
cell kill, reactive
ox-
the early phase after transplantation, whereas other functions are normal or even enhanced. These latter nonspecific defense mechanisms are presumed to be of crucial significance for survival.
MATERIALS Mice
AND METHODS
Male 6-8-week-old C57B1/6 (H-2”) mice were received from burg, S#{252}lzfeld,Federal Republic Cell
(H2b) and Charles River, of Germany.
DBA/2 W#{252}rz-
Lines
YAC-l,
a Moloney-virus-induced lymphoma of A/Sn P815, a mastocytoma induced by methylcholanthrene in DBA/2 mice; EL-4 a benzopyrene-induced lymphoma of C57BL/6 origin; and the fibroblast cell line
origin;
Received
January
6, 1989;
accepted
February
24,
1989.
Reprint requests: Joachim Roesler, Department of Immunobiology, Fraunhofer-Institute for Toxicology and Aerosol Research, NikolaiFuchs-Str. 1, 3000 Hannover 61, Federal Republic of Germany.
Mos Early After Bone Marrow Transfer
L-929 of
C3H
origin were maintained in RPMI 1640 medium supplemented serum (FCS) and 0.1% gentamicin Grand Island, NY, hereafter referred
as with (all to as
tissue cultures 10% fetal calf from Gibco,
complete
me-
dium).
Antisera Alloantisera against H-2’’ cells were produced by injecting DBA/2 (H2d) mice with 30 x 106 EL-4 tumor cells (H-2”). The mice were boosted with a second inoculum of EL-4 cells (1 x 1 06), and the immune sera were collected 5 days after boosting. The same protocol was used to obtain antiH2d alloantisera from C57B1I6 mice (H_2b) immunized with P815 (H2d) tumor cells. No cross-reaction was observed between the two antisera obtained in this way. A 1:100 dilution of each antiserum caused the complete lysis of the cells bearing the respective H-2 antigen in the presence of a 1:6 dilution of Low-tox-M rabbit complement, lot (Cedarlane, Hornby, Ontario). Antiserum against sheep red blood cells (SRBC) was taken from C57 mice, which had been injected intravenously (i.v.) with ml of a 2% SRBC suspension and boosted 4 days later.
4353
0.5
Preparation
of L-Conditioned
Medium
L-929 cells (1 x 106) were seeded in petri dishes in 10 ml of RPM! + 5% FCS. After 24 hr the adherent cells were washed with serum-free RPM!, and fresh incubation medium was added. After a multiplication period of about 4 days, the cells had formed a monolayer across the bottom of the dish. The medium was then aspirated and assayed for content of macrophage colony-stimulating factor (M-CSF) by its capacity to induce macrophage colonies in bone marrow cultures [6]. To achieve this, 10% L-929 supernatant was usually highly efficient. The L-929 conditioned medium used for triggering cell growth consisted of 30% L-929 supernatant in complete medium. Preparation
of Chimeras
Male DBA/2-mice, 7-9 weeks old, were used as bone marrow recipients. They were irradiated by a 60Co source in plastic boxes beneath a 5 mm-thick plastic glass cover, to avoid build-up effects in the mice. Dosage was controlled by entrance and exit measurements on five mice, using thermoluminescence dosemeters (lithium fluoride crystals) fixed to the back and abdomen of each mouse. The mean entrance dose was 10.22 Gray ± 0.15 (68% confidence level) at an intensity of 1.2 Gray/mm; the exit dose was 9.46 ± 0.3 Gray. The measurements were kindly performed by Dr. K. Rittmann, a physicist at the Medizinische Hochschule, Hannover. The dose was chosen to minimize irradiation side effects
135
and reversion of bone-marrow-derived cells to the recipient haplotype. Two hours after irradiation, the mice were injected i.v. with 5 x 106 bone marrow cells from C57b116 donors. This bone marrow had been pretreated with monoclonal anti-Thy 1.2 antibody (Cedarlane, Ontario, Canada) as described [9,14]. Survival rate was about 70%, without antibiotics in the drinking water, under conventional conditions in a large colony. Signs of GVHD or wasting disease were never observed; the mixed lymphocyte reaction of chimeric splenocytes against irradiated (20 Gray) splenocytes of the recipient haplotype was always negative. Animals used as controls were always untreated age-matched DBAJ2 and C57BI/6 mice.
H-2 Typing This was performed by microcytotoxicity testing. Briefly, spleen cells of the chimeras were suspended in medium without serum (2 x l0 cells in 150 1.il), supplemented with anti H-2 antiserum (1:50) and complement (1:20), and incubated for 2 hr at 37#{176}C. Cell lysis was established by trypan blue dye exclusion. In each typing assay, positive and negative control cells were included to confirm the specificity of the antisera treatment and the activity of the complement. All tests were carried out in triplicate.
Preparation of Spleen Cell and Liver Nonparenchymal Cell (NPC) Suspensions Spleen cell suspensions were obtained by standard techniques and depleted of erythrocytes by hypotonic lysis. The pronase method, as described by Knook and Sleyster [5], has been used with minor modifications for isolation of liver NPC. In brief, livers of pentobarbitalanesthetized mice were blanched by perfusing with 10 ml of HEPES-buffered Hanks’ balanced salt solution (HBSS), pH 7.4, via the portal vein, followed by perfusion with 5 ml of pronase E (Boehringer, Mannheim, Federal Republic of Germany) solution (0.2% in HBSS). The livers were excised, minced, and incubated for 45 mm at 37#{176}C, with constant stirring, in 40 ml of 0.15% pronase E in HBSS. The resulting cell suspension was washed twice in cold Dulbecco’s minimal essential medium (DMEM) and subjected to metrizamide gradient centrifugation. NPC from the interface were washed twice in serum-free DMEM and finally resuspended at 5 x l0/ml for further separation. Viability of NPC after metrizamide gradient centrifugation was > as judged by trypan blue dye exclusion.
95%,
Separation
of Macrophages
(M4)
Alveolar Mcj were harvested by cannulating chea of exsanguinated mice with a catheter mm) (Deutschel Abbott, Wiesbaden, Federal
the tra(22 by 32 Republic
136
Roesler et al.
of Germany) and flushing the lungs with 2 ml of cold heparinized medium. The last procedure was repeated five times. Cells were then further purified by selective adherence to plastic. Liver M (Kupffer cells) were obtained from the NPC suspension by selective adherence. Splenic M were obtained by applying spleen cell suspensions to a discontinuous Percoll gradient that consisted of four steps from 30 to 60% in 10% steps. Cells from the first two layers were harvested and further purified by selective adherence. Thioglycolate-induced peritoneal cells were obtained from animals that had been injected i.p. with 3 ml of 3% thioglycolate broth 24 hr earlier. These cells were harvested by lavage of the Pc with 5 ml of cold RPM! medium containing 10 U/ml of heparin. The time of 24 be, instead of the more conventional 72 hr, was used to get information about cell recruitment at different precise time intervals after bone marrow transfer.
Cell Staining Cells were stained for microscopic analysis with MayGrUnwald-Giemsa solution according to Pappenheim [e.g., 11] and controlled by nonspecific estherase staining [4]. Phagocytosis
In Vitro
Ingestion of native SRBC and formation of rosettes (with three SRBC or more) were employed as indicators of spontaneous phagocytosis. Ingestion and rosette formation of SRBC sensitized with heat-inactivated mouse serum containing anti-SRBC IgG were used for evaluation of Fc-receptor-mediated phagocytosis. M4 were preincubated to permit adherence to Falcon plastic dishes (alveolar M and Pe-M, 4 hr; spleen-derived M, 14 be; and liver-derived M4, 2 days), washed, and incubated with SRBC for 2 hr at 37#{176}C (5% CO2, M4:SRBC ratio = 1:10). After washing twice to remove free erythrocytes, the cells (at least 200) were counted by light microscopy, and the percentage of positive cells was evaluated. For simplicity phagocytosing and rosetteforming cells were added together, each contributing about to the result.
50%
Measurement of Luminol-Dependent Chemiluminescence, CL A total of 2 x l0 cells cultured in HEPES-buffered complete medium was used for measurement of luminoldependent chemiluminescence. A 15 p.1 amount of luminol (5-amino-2,3-dihydro1 ,4-phthalazinedione; Boehringer, Mannheim, Federal Republic of Germany) was added at 2 mg/mI, and the background CL was monitored in a six-channel Berthold Biolumat [1]. When background activity had reached constant values, about 108 zymosan particles were added. CL resulting from
subsequent generation of reactive oxygen intermediates (RO!) was then measured for 30 mm. Software for computerized calculation of peak activities and integrals was supplied by Berthold (Wildbad, Federal Republic of Germany).
Macrophage
Activation
Cells in 96-well flat-bottomed microtiter plates were incubated for 18 hr before the assay in the presence of recombinant gamma-interferon (rIFN-’y; Genentech; kindly supplied by Boehringer, Ingeiheim, Federal Republic of Germany) at a concentration of 102 U/ml, plus 10 ng/ml of Escherichia coli lipopolysaccharide (LPS) (Difco, Frankfurt/M., Federal Republic of Germany), as previously described [10]. Before the assay, the supernatant was removed and replaced with fresh complete medium.
Measurement of Tumor (TNFa) Activity
Necrosis
Factora
TNFa activity in culture supernatants of M was determined in a biological assay on actinomycin-D-treated L-929 cells, as described [12]. The units given are the reciprocal values of the supernatant dilution that would cause lysis of of the L-929 cell layer.
50%
Proliferation
Assay
([3H]-dThymidine) incorporation into replicating cells was measured to determine the proliferative ability of different cell populations, 5 x l0 cells/well in complete medium were seeded into wells of flat-bottomed microtiter plates and incubated either in the presence or in the absence of 30% L-conditioned medium at 37#{176}C for 3 days. One microcurie p.Ci/well of [3H]dThd was then added, followed by another 18 hr incubation period. The cells were then harvested by a Skatron suction filtration apparatus. The 3H-radioactivity bound to the fiberglass filter was determined by 3-scintillation counting.
51Cr Release
Assay
Against
Tumor
Cells
Effector cells in triplicate microtiter wells were incubated with 5 x l0 5tCr-labelled (200 p.Ci for 1 hr at 37#{176}C in 5% C02) tumor cells at 37#{176}C, 5% CO2. At the end of the incubation period, an aliquot of the supernatant was collected, and its radioactivity was measured in a -y-counter. The spontaneous release (SR) was determined by incubation of tumor cells without effector cells and never exceeded 25% of the total radioactivity incorporated by the target cell (cpmto,). The percentage of specific lysis was then calculated according to the formula: % spec.
lysis
=
cpmexp.
-
cpmtot
SR
X
100%.
Mos Early After Bone Marrow
Transfer
137
5 L
a
‘I
3
2
1
o
1
2
4
3
5
6
7
8
9
10
11 12
13
days
14 after
15
16
17
18
19 20
21
IrradIation
Fig. 1. White blood cells (WBC) after bone marrow transfer. Myelold cells (--); lymphocytes (-+-); WBC of DBA that were irradiated but received no bone marrow (_*_); ten animals per group; two animals were tested every day and the results averaged; time intervals
for further analysis
Listerla
().
monocytogenes
This organism (strain EGD) was kept virulent by continuous mouse passage. Cultures were obtained by growing a sample of spleen homogenate from an infected mouse in trypticase-soy broth. Bacterial suspensions were appropriately diluted in 0.9% NaC1 and injected i_v. in a volume of 0.5 ml. The number of L. monocytogenes injected was confirmed by plate counting. Fortyeight hours after infection the spleen and liver of the infected animals were homogenized, and the resulting number of colony-forming units (CFU) was determined again by plate counting. Statistical
Analysis
Most results are given as arithmetic means ± standard deviation. TNF units are given as geometric means. For statistical analysis of the data, Wilcoxon’s rank test was used. Differences were considered significant according to the P values indicated.
RESULTS We evaluated the extent of repopulation of the various organs of our bone marrow chimeras. Figure 1 shows the time course of the increase in the white blood count (WBC) after transplantation. As expected, the number of lymphocytes decreased rapidly after irradiation, followed by a decrease of polymorphnuclear cells (PMN) on day 3. Control mice that received no bone marrow died between days 9 and 11. Trans-
planted animals already showed an increase of white blood cells on day 6, accompanied by a high percentage of mature and immature myeloid cells in the peripheral blood, indicating a good homing of myeloid cells in the allogeneic environment. We chose two time periods for more detailed analyses of leukocyte number and function in spleen, liver, lung, and peritoneal cavity (Fig. 2). An initial celullarity was determined by analysis on days 3, 4, and 5. The first stage of normalization of the white blood count was analyzed on days 8-12. In Table 1 we only listed the number of M. PMN always lay in the same range (not shown). The remaining white cells were immature myeloid cells and lymphocytes.
Days 3-5 After
Transplantation
M4) in spleen, liver, lungs, and peritoneum. As expected, the minimum white blood count was accompanied by a sharp decrease in the number of splenocytes (Table 1). The ratio of M to other leukocytes was increased by their higher resistance to irradiation, although the absolute number of M4 was decreased compared with controls. These M were of both donor and recipient origin in very fluctuating percentages. They were functionally not impaired, showing normal phagocytosis of SRBC, which could be increased by addition of mouse serum containing a-SRBC-IgG (Table 2). Lung and liver also showed a decrease in the number of M, which nevertheless retained their normal ability to phagocytose.
138
Roesier
et al. Determination
of listerial
Infection
of DBA/2
rradiation xl0exp6 C57Bl/6
days
with
0
Isolation marrow, Fig. 2.
After
Flow
Bone Marrow
chart
Days after transfer
Spleen
3-5 8-12 Controls’
LiverNPC
Yie1d
of cells
bAverage
after
of ten
±
33.0”
±
35.0
(%)
leukocytes
0.26
±
0.1
26
14.00 5.60
±
8.1
25
±
2.7
7
2.4 1.4
44 43 43
1.36±0.3 5.37
±
5.7
±
3.0
2.44
±
0.15 0.08 0.28
±
013b
0.13
± 0.12
>85
±
0.066
0.07
±
0.06
>85
±
0.20
0.24
± 0.10
>85
3-5
1.1
±
08b
0.33
±
0.24
30
97
±
28b
4.10
±
1.20
Controls’
18.6
±
7.6
6.20
±
2.50
42 33
preparation.
2. Macrophage
(M’)
the cells
Phagocytosls
were
of SRBC
harvested.
After
Days after transfer
Spleen
Bone
Marrow
Spontaneous
3-5 8-12
Peritoneal
106)
chimeras.
M4 origin
Lung
04b
(X
8-12
‘Results of 5 C57 and 5 DBA were averaged. dmioglycolate was injected i.p. 24 hr before TABLE
±
M4 among
No. of M4
3.1 +066 12.4 ± 56b
3-5 8-12 Controls’
Pe-M4s (thioglycolate induced)d
Organs
10)’
1.0 57.0 85.0
Controls’
Cavity
design.
leukocytes
of
(X
3-5 8-l2
M4,)
peritoneal
tests
in Different No.
origin
from
spleen,
of experimental
Transfer
Macrophage (M4)
1’l
cells
of
liver,
lung,
for In vitro
Lungs (alveolar
with cells
;
2
1. Cellularity
listens
origin
bone
TABLE
and livec
in spleen
5xlOexp3
mice; reconstitution treated bone marrow
antl-Thyl,2
CFU
Transfera After preincubation of SRBC with lgG (%)
(%)
b b
126±
35 N.S. 48 N.S.
154 152
b 12,P
