Major Histocompatibility Complex Class 11

Major Histocompatibility Complex Class 11-Mediated Inhibition of
Hematopoiesis in Long-Term Marrow Cultures Involves Apoptosis and
Is Prevented by c-kit Ligand
By Dae-Sik Hong, Cassandra Beckham, Ralf Huss, Jong Wook Lee, David Hockenbery, Jeffrey A. Ledbetter,
and H. Joachim Deeg
Expression of major histocompatibilitycomplex (MHC) class
II molecules is developmentally regulated and lineage dependent. Their role in hematopoiesis is not well defined.
Previous studies in a canine model showed thatdogs given
920 cGy of total bodyirradiation. transplanted with autologous marrow, and treatedwith anti-MHC class II monoclonal
antibody (MoAb) immediately posttransplant experienced
only a transient granulocyte recovery that was followed by
graft failure. In the present study, the effect of anti-MHC
class II MoAbs oncanine in vitro hematopoiesis was investigated. Anti-MHC class II MoAb H81.9 or B1F6 (both recognizing nonpolymorphic determinants) had no inhibitoryeffect
when added directly t o colony-forming unit-granulocytemacrophage (CFU-GM) grown in agar. However, the addition of intact MoAb or as F(ab’), fragments t o long-term
marrow cultures (LTMCs) resulted in a dose-dependent inhibition of the generation of CFU-GM among nonadherent
profound with MoAb added at the
cells. Inhibition was most
time of initiation of culture. However, even if MoAb was
added 3 weeks after rechargingLTMCs. CFU-GM generation
rapidly decreased. In addition, the numberof adherent cells
in LTMCs decreased;predominantly fibroblast-like cells with
prominent cytoplasmic vesiculation remained. Acridine orangelethidium bromide staining and TdT-mediated deoxyuridine triphosphate-digoxigenin nick end labeling {TUNEL)
tests showedan increase inthe proportionof apoptotic cells
in both thenonadherent and adherent compartments. Binding ofanti-MHC class II MoAb t o unfractionated marrow cells
resulted in an increase in free (Caa’)i; no changes in tyrosine
phosphorylation patternwere observed. The additionof
stem cell factor (SCF), but not granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating
factor, t o LTMCs prevented apoptosis, and the generation
of CFU-GM was indistinguishable from controls. Similarly,
a supportive adherentlayer was maintained. Thus, anti-MHC
class II MoAbs interfere with hematopoiesis both in vitro
and in vivo. The mechanism involvesprogrammed cell death
in subpopulations of adherent and nonadherent
cells. Inhibition of hematopoiesis is abrogated by exogenous SCF.
0 1995 by The American Society of Hematology.
T
class I1 MoAb immediately posttransplant (days 0-4) show
HE GENES and gene products of the major histocomonly a transient increase in granulocytes after the postirradiapatibility complex (MHC), termed HLA in humans’
tion nadir, which is followed by graft failure and marrow
and DLA in the dog,* play a central role in the regulation
aplasia.*>These findings suggest that MHC class 11’ cells
of immune responses, immune-mediated diseases, and
either with hematopoietic or accessory function and required
transplantation. The role
of these genes in hematopoiesis
is less
for sustained hematopoietic recovery, are inhibited by the
well defir~ed.~.~
Although class I antigens are widely expressed
administration of anti-MHC class I1 MoAb. Alternatively,
on lymphohematopoietic cells,the expression of MHC class 11
MHC class 11-mediated signals may interfere with hematois restricted, developmentally regulated, and inducible in a tispoiesis via downregulation of hematopoietic growth factors
sue and cell lineage-dependent fashion.” Human hematopoior induction of negative regulators of hematopoiesis.
etic progenitor cells, including colony-forming unit-granulocyte-macrophage (CFU-GM), burst-formingunit erythroid, and
Long-term marrow culture (LTMC) systemsz4 provide
colony-forming unit granulocyte, erythroid, monocyte, megauseful in vitro models of hematopoiesis. These cultures are
karyocyte, express HLA-DR and -DP, whereas HLA-DQ anti- dependent on the establishment of a complex adherent layer
gens are la~king?~”’”~
Maturation of granulocytes is associated
composed of both hematopoietic precursor cells and stromal
with a loss of cellsurfaceexpression of class I1 antigens,
cells that simulate the marrow microenvironment?‘2h With
whereas expression is maintained on functionally mature monocytes and macrophages?,’’ The current notion is that among
From the Clinical Research and Molecular Medicine Divisions,
adulthumanmarrowcells,theearliestidentifiableprecursor
Fred
Hutchinson
Cancer Research Center; and Bristol-Myers
cells are CD34TD38- lin- c-kit+ DR-.“.I6 However, depenSquibb, Inc, Seattle, WA.
dent upon culture conditions, a greater yield of long-term culSubmitted October 13, 1994; accepted June 26, 1995.
ture initiating cells maybe derived from DR’ than DR- precurSupported in partby
grants CA18221, CA31787, CA15704,
sors.” In addition, Traycoff et a l l 8 have shown that long-term
HL36444, and DK42716 from the National Institutes of Health,
culture-initiating cells in human cord bloodare CD34’ DR’.
Department of Health and Human Services. D X Hong was supIn some species such as the dog” or guinea pig:” MHC
ported by a grant from Soon Chun Hyang University, Seoul, Korea:
R. Huss by the Deutsche Forschungsgemeinschaft, Bad Godesberg,
class I1 molecules are expressed rather broadly including,
Germany: and J.W. Lee by Catholic University Medical College,
eg, resting T cells. In vitro cytolytic treatment of canine
Seoul, Korea.
marrow with the murine anti-MHC class I1 monoclonal antiAddress reprint requests to H. Joachim Deeg, MD, Fred Hutchinbody (MoAb) 7.2 before autologous transplantation prevents
son
Cancer Research Center, 1124 Columbia St, M318, Seattle, WA
hematopoietic reconstitution in lethally irradiated dogs.”
98104.
Conversely, positively selected (7.2’) marrow cells are capaThe publication costs of this article were defrayed in part by page
ble of sustained hematopoietic reconstitution.” In more recharge payment. This article must therefore be hereby marked
cent studies, we manipulated MHC class I1 gene products
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
in vivo: Dogs given 920 cGy of total body irradiation and
indicate this fact.
autologous marrow uniformly recover normal hematopoie0 1995 by The American Society of Hematology.
sis; however, dogs that, in addition, are given anti-MHC
0006-4971~95/8609-0006$3.00/0
Blood, Vol86, No 9 (November l), 1995:pp 3341-3352
3341
HONG ET AL
3342
Table 1. Addition of MoAbto CFU-GM Assay
MoAb Added to Assay'
H81.9
61F6
116 2 12
278 2 21
110 2 16
250 2 18
118 2 20
256 2 19
12 2 3
-
-
Experiment
None
~~
1
2
3 pretreatment of cells with
H81.9 + C ' t
~
* Marrow-derived
mononuclearcells (1 x lo5)were plated per CUIture; each assay wasperformed in triplicate and colonies were
counted on day 14. MoAb concentrations were those found to be
effective in long-term cultures, H81.9 at 10 pglmL and B1F6 at 100
pglmL.
t Cells were incubated at 4°C with H81.9 for 1 hour, washed and
then incubated with complement(C') for 1 hour, washed, and plated.
this support, fresh marrow cells recharged onto this layer
are able to generate mature hematopoietic cells for extended
periods of time. We have used such a system in an attempt
to define the mechanisms by which anti-MHC class I1
MoAbs may interfere with hematopoiesis, and show that
these MoAbs inhibit hematopoiesis from nonadherent cells
in LTMCs and morphologically alter the adherent layer.
MHC class 11-mediated inhibition is abrogated by the addition of exogenous c-kit ligand or stem cell factor (SCF).
MATERIALS AND METHODS
MOAhs
MoAb H81.98.21 (H81.9; IgG2a), generated against mouse MHC
class 11 antigens, i s cross-reactive with human HLA-DR and canine
H 81.9 F( ab72
B
3
1
4
WEEK
WEEK
B1F6
C
- .
"....
~
"l
D
G3G6
A
I
1
?
1
WEEK
WEEK
fig 1. Dose/response effect of anti-MHC dass II MoAbs on the generdonof C N G M from LTMCs. LTMCs, after recharging, received one of the
following MoAbs: (A) MoAb H81.9, (B) MoAb H81.9 F(ab'1, (C) MoAb BlF6, and (D) G3G6. Results are expressed as percentage of colony formation
2 SD in control cultures. Absolute numbers for C N G M in control cultures at weeks 1,2,3, and 4 were (A) 123 2 13,72 2 4,31 2 4, and 16 2 2; (B)
90~3,62~4,38~4,and18~2;(C)99~5,52~6,36~2,22f1and1122;and~D~99~9,52~5,3628,and22~4,respectively.
MHC CLASS II-MEDIATED INHIBITION OF HEMATOPOIESIS
3343
H 81.9 1Oug/ml
time difference
-...................................
............................
Fig 2. Time-dependent effect of MoAb H81.9 on
the generation of CFU-GM from LTMCs. MoAb H81.9
(10 pg/mLI was added to flasks at initiation of cultures, at the time of recharging, or 6 days after recharging. Results are expressed as percentage of colony formation ? SD in control cultures. The average
numbers for CFU-GM in control cultures at weeks1,
2.3, and 4 were 123 ? 16,69 ? 6,29 ? 2, and 16 i:
2, respectively.
+ after I. e h .
1
MHC class I1 framework determinant^."^'^ B I F6 (IgG2a) recognizes
canine MHC class I1 framework determinants and is cross-reactive
with HLA-DR and -DP.” F(ab’)’ fragments of H8 I .9 were obtained
by digestion withpepsinandpurificationoverprotein-A
columns
as described.’” MoAbG3G6 (IgG2a). specific for the human plateletassociatedglycoprotein lIb/llla (C. Badger,unpublishedobservations, 1994) and nonreactive with canine cells, was used as control.
MoAbs were purified from murineascites or obtained from supernatant of hybridoma grown in the Fred Hutchinson Cancer Research
Center bioproductionfacility. The MoAbswerecharacterized
by
standard techniques and shown to
be free of mycoplasma contamination as described.”
2
3
4
Week
scribed.” MNCs, 7.5 X IO4, were cultured for 14 days at 37°C in a
humidifiedatmosphere of 5% CO, in air in 35-mmPetri dishes
containing 2 mL of agar medium. The agar medium consisted of an
equal volume mixture of 0.6% (wthol) Bacto agar (Difco, Detroit,
MI) anddouble-strengthDulbecco’smodifiedEagle’smedium
(GIBCO, Grand Island, NY) containing 40% (vollvol) heat-inactivated prescreened human AB plasma. Three replicate cultures per
testwereassayed.
The morphologyof cells in thecolonieswas
determined on cytospin preparations of individually picked colonies
stained accordingto Wright-Giemsa (Sigma, St Louis, MO).In addition, fresh marrow MNCs, either unmanipulated or incubated with
MoAb for 30 or 60 minutes before being placed in culture, were
assayed for CFU-GM.
LTMCs
Bone marrow (BM) aspirates were obtained from the humerus of
anesthetizednormal dogs, and LTMCswere established as described.“ Briefly, mononuclear marrow cells (MNCs; 2 X IO’) were
cultured in 25-cm’ tissue culture flasks (Costar, Cambridge, MA) in
RPMI-I640 medium (MA Bioproducts, Walkerville, MD), supplementedwith20%prescreenedheat-inactivatedhorseserum,
IO-’
moln hydrocortisone91 phosphate (Sigma Chemical CO, St Louis,
MO), I % nonessentialamino acids, I % pyruvate, 2% glutamine,
and 1% penicillin-streptomycin. Cultures were maintained at 37°C
in a humidified atmosphere of 5% CO, in air. After I week, nonadherent cells were removed, and halfof the spent medium plus an
equalvolumeoffreshmediumandfreshlyaspiratedautologous
marrow buffy coat cells were recharged onto the adherent layers.
Anti-MHC class I1 MoAb was addedinto flasks either upon initiation
of culture or at the time of recharging. In some experiments, MoAb
was added to cultures 6 days after recharging, ie, 24 hours before
assaying nonadherent cells for colony formation in semisolid agar.
Starting I week after recharging, nonadherent cells were procured
at weekly intervals, pelleted, and counted, and aliquots were assayed
for CFU-GM. The remaining cells were returned to the long-term
culture flasks with spent medium and fresh medium mixed
I :I.
CFU-GM Assay
CFU-GM assays wereperformedwithnonadherent
cells from
LTMCs and inadditional experiments with fresh marrowcells. Nonadherent cells fromLTMCswereassayed
for CFU-GM as de-
Hematopoietic Growth Factors
Recombinant canine c-kif ligand (stem cellfactor; rcSCF), rc granulocyte-macrophage colony-stimulating factor (rcGM-CSF), and rc
granulocyte colony-stimulating factor (rcG-CSF) were provided by
Amgen Inc (Thousand Oaks, CA). These factors were used at concentrations determined in ancillary studies: SCF at 1 to 500 ng/mL,
GM-CSF at 10 to 1 0 0 nglmL, and G-CSF at 1 0 0 to 500 ng/mL. In
various LTMC experiments, either one of these factors was added
alone or in conjunction with anti-MHC class I1 MoAb with the aim
of determining whether the MoAb-mediated effect was abrogated.
Morphologic Studies on LTMC
Lighr microscopy (LMJ. In additiontostandard
culture flasks,
LTMCs were established on chamber slides in flaskettes with I-cm’
wells (Nunc, Naperville, IL), as described.’> After I week in culture,
the nonadherent cells were removed, and fresh autologous marrow
buffy coat cells were recharged onto the adherentlayers as described
above, andanti-MHC class I1 MoAbwasaddedatvarioustime
intervals. The adherent stromal layers were examinedby LM. Additional slides were stained with acridine orange and ethidium bromide
or examined by TdT-mediateddeoxyuridinetriphosphate-digoxigenin nick end labeling (TUNEL; Apop Tag Kit; OnCor Inc, Gaithersburg, MD) test to examine for apoptotic changes (see below).l4
Electron microscopy. Canine marrowMNCswereprocured as
described on chamber slides in flaskettes with I-cm’ wells and cultured in the presence of anti-MHC
class I1 MoAbs. Stromal cells
HONG ET AL
cipitation of phospholipase C (PLC) yl wasperformed as described.'" Immune complexes were collected on protein A-sepharose
beads, washed, subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis.'5 lmmunoblots with anti-PLCy1 and antiphosphotyrosine were performed and antibody binding detected using
'"l protein A and autoradiography as described."'."
Measurement of Cytoplasmic Calcium Ion [(Ca")i]
Concentration
Ca"ion flux in indo-l (Molecular Probes, Eugene, OR) loaded
cells was measured with a model 50 HH12150 flow cytometer (Ortho,
Westwood, M A ) as described.'"'" Briefly, canine mononuclear cells
were loadedwith indo-l acetoxymethyl ester by incubation for 5
minutes ar 3 7 T, followed by incubation on ice at 4°C. Cells were
thenwashedand resuspended in fresh mediumand stored in the
dark on ice until analysis. The modification of maintaining cells at
4°C rather than37°C or room temperature. as is customary with
Fig 3. Loss of adherent stromal layer in MoAb H81.9-treated cultures. MoAb H81.9 110 pg/mL) wasadded either alone or alongwith
SCF t o flaskettes (l-cm* wells) after
recharging. Typical areas of each
culture are shown (x10or x20): (A) control, (B)MoAb H81.9, and (C)
MoAb H81.9 plus SCF (10 ng/mL). AlthoughH81.9-treated flaskettes
show a loss of adherent cells relative t o controls, the addition of
SCF concurrently with H81.9 prevented adherent cell loss; in fact,
adherent cells appeared larger and showeda tendency toward cluster formation.
were fixed in Kamovsky'slKamosky2, washed in 1% OsO, for I
hour on ice, andthen dehydrated in sequential ethanol series and
embedded. The sections were placed on copper grids and stained
with uranyl acetate and lead citrate, and examined on a JEOL 1 0 0
SX electron microscope."
Immunoprecipitation and lmmunoblots
Cells were lysed on ice with NP-40 lysis buffer and centrifuged
at 13,OOOg to remove insoluble material as described.35Immunopre-
Fig 4. Electron microscopic appearance of stromal layer. Cells
were maintained in complete medium with (B) or without (A) addition of MoAb
H81.9 at 10 pg/mL. Cultures were photographed7 days
after MoAb addition. After MoAb treatment, there was an increase
in fibrotic bands and vesiculation in the cytoplasm.
ASS
OF HEMATOPOIESIS
MHC
3345
H 81.9
B
+ SCF
1 - 1021
H 81.9+SCF Monglml
-2
120
H 81.9+SCF lOOng/ml
100
H 81.9+SCF lOng/ml
-E r n
H 81.9+SCF 5ng/ml
H 81.9+SCF lng/ml
'pM)
3
240
U
0
81.9
m
+ SCF
H 81.9 +G-CSF
81.9 + GM-CSF
0
1
7
3
WEEK
'0
>
I
1
2
3
4
5
WEEK
Fig 5. Effect of rcSCF, rcG-CSF, or rcGM-CSF on the generation CFU-GM
of
in MoAb H81.9-treated LTMCs. LTMCswere culturedin complete
medium and treatedas indicated. (A) MoAb H819(10 pg/mL) alone, or MoAb H81.9 plus SCF (10 ng/mL), G-CSF (100 ng/mL), or GM-CSF (10
nglmLl were added at the time ofrecharging. (B) LTMCs were performed in the presence of MoAb H81.9 (10 pg/mL) plus SCF (at l,5, 10,
100, or 500 ng/mL) bothadded at the timeof recharging. Colony formation from nonadherent cells 2 1 SD is expressed as percent of control
cultures. The average numbers of CFU-GM in control cultures at weeks 1, 2, 3,4, and 5 were as follows: (A) 87 ? 7,55 f 4, 35 f 2, 20 2 3,
and 14 2 1; (B) 116 f 12,76 c 7,48 f 3,19 2 2, and 9 2 2.
human ceIls,29.4(1
was necessary to prevent rapid turnover and leakage
of indo-l from cells. For each assay, indo-l loaded cells were diluted
to I X I06/mL with medium, equilibrated at37°C in a waterbath,
and analyzed by flow cytometry. Histograms were analyzed in regard
tothemean indo-l violetblue fluorescence ratio as a function of
time, and the percentages of cells with a particular indo-l ratio above
the mean for control cells was determined.2s.29~J"
Microscopic Determination of Apoptosis in Nonadherent
and Adherent Cells of LTMCs
Viability of nonadherent and adherent cells was determined using
trypan blue dye exclusion and acridine orange/ethidium bromide
CY
Y.
staining. Cell morphology was evaluated on
May-GriinwaldGiemsa-stained cytocentrifuged cell preparations. Apoptotic cellular changes were identified as described considering cell membrane
alterations, chromatin condensation, and nuclear fragmentation?'.''
In addition, the TUNEL test was appliedto anti-MHC class I1
MoAb-treated nonadherent and adherent cells. Cultured nonadherent
cells and adherent cells detached by treatment with trypsin (0.05%;
S minutes) were centrifuged for 4 minutes at 4 0 0 ~
at room temperature. The supernatant was discarded, and the pellet was resuspended
in the remaining medium. Four percent formalin was added to the
suspension. Drops of the cell suspension were placed on slides precoated with 0.01% poly-L-lysine and air dried. TdT labeling was
performed as described by Gavrieli et a1,'4 except that peroxidase
was developed with diaminobenzene. Slides were counterstained
with methyl green.
Flow Cytometric Analysis of Apoptosis in LTMCs
Min. '2 5"2 5"2 5.-25 . . 2 5 2 5 2 5 2 5 2 5
"-SF
W-
7
.
Fig 6. lmmunoblot of marrowmononuclear cell lysates with antiphosphotyrosine. Cells were exposed t o various MoAbs for 2 or 5
minutes, and processed as described under Materials and Methods.
No change in phosphorylation pattern was observed. Avidin, avidin
control. The abbreviations represent various anti-MHC class II
MoAbs: H,H81.9;B,
BlF6; Ca.Ca1.41; F(ab')>, fragments of H81.9;
HE, HBlOa; p4, p4.1.
To further quantitate anti-MHC class 11 MoAb-mediated
apoptosis, a flow cytometric analysis ofpropidium iodide (PI)stained cells was used with modifications as described?' After various incubation times after recharging in LTMC, nonadherent and
adherent cells were washed and pelleted, and the pellet gently resuspended in I mL hypotonic PI solution (Sigma ~ 1 3 0 4 1, 0 0 &mL;
0.I %
' sodium citrate, 0.1% Triton X- 100; Ribonuclease A: Sigma R
4875.0. I mg/mL). Samples were allowed to equilibrate for at least I
hour in the dark before analysis. Fluorescence analysis of individual
nuclei was performed with the use of a FACScan flow cytometer
equipped with an argon laser at 488 nm and 250 mW light output and
lysis I1 software (Becton Dickinson, San Jose, CA). The fluorescence
intensity from cell nuclei stained with PI is proportional to the cellular DNA content. Events ( I O X IO4) were collected, stored, and
analyzed by Multicycle software developed byDrP.
Rabinovitch
(University of Washington, Seattle, WA).
Transmission Electron Microscopy of Apoptosis in LTMCs
Cultured nonadherent and adherent cells were collected by
scraping with CellScraper (Baxter, McGaw Park, IL) in the presence
of MoAb H81.9 alone or MoAb H81.9 plus SCF, andwashed in
phosphate-buffered saline (PBS). After washing, the cells were fixed
3346
HONG ET AL
*.ol+
2.5 t
0
5
10
Time (min)
10
Time (min)
in Karnovsky’slKamosky2 for 1 hour on ice. The cells were rinsed
in PBS, postfixed with 1% Os04 for 1 hour on ice, and then dehydrated in sequentialethanolseries and embedded in Polybed 812
(Polysciences, Inc, Warrington, PA). The sections were placedon
copper grids and stained with uranyl acetate and lead citrate, and
examined on a JEOL 100 SX electron microscope as de~cribed.~~
RESULTS
In Vitro Hematopoiesis is Inhibited by Anti-MHC Class II
MoAbs
Incubation of MNCs with MoAb H81.9 or BlF6 for 30
minutes or 1 hour before being placed in culture didnot
effect CFU-GM colony formation; only pretreatment of cells
with MoAb plus complement reduced the number of CFUGM (Table 1). However, if MoAb [intact or as F(ab’), fragments] was added to LTMCs, the ability of nonadherent cells
to form CFU-GM was inhibited in a dose-dependent fashion
(Fig 1). Inhibition was most profound with MoAb added at
the time of initiation of cultures and less so when MoAb
was added later (Fig 2 ) . Considering the possibility that the
early presence of MoAb would not allow for the establishment of a functional adherent layer and normal contact to
nonadherent cells, an adherent layer was established, recharged with nonadherent cells 1 week later, and cultures
were left unperturbed for 2 weeks before H81.9 (10 pg/ml)
was added. Beginning again l week later, nonadherent cells
were assayed for CFU-GM. As with the earlier addition
of MoAb, CFU-GM formation decreased to unmeasurable
numbers by 3 weeks (not shown).
Anti-MHC Class I1 MoAb-Mediated Loss of Adherent
Layers
As shown in Fig 3, in the presence of MoAb H81.9 there
was a progressive decrease in the density of adherent cells
in LTMCs. By light and electron microscopic examination,
remaining cells showed fibroblastoid characteristics. Furthermore, adherent cells in MoAb-treated cultures contained
more prominent cytoplasmatic vesicles than did cells in nontreated cultures (Fig 4).
Effect of Recombinant Growth Factor on Anti-MHC Class
I1 MoAb-Mediated Inhibition of LTMCs
Previous studies had shown that dogs treated with MoAb
H81.9 were rescued from graft failure if treated with exoge-
Fig 7. Calcium mobilization in
MoAb
H81.9treated marrow mononuclearcells.Mononuclear
cells were loaded with indo-l, asdescribedunder
Materials and Methods. Biotin conjugated MoAb
H81.9 was added and cross-linked by avidin at the
time point indicated[arrow). The horizontal axis indicates real time progression; the vertical axis indicates the violet/blue ratio, an indicator of free calcium (A) and the proportion of cells responding (B).
nous SCF concurrently with MoAb administration, even
though the kinetics of hematopoietic recovery were not completely normal?’ Therefore, we attempted to characterize a
comparable effect of SCF (or other growth factors) in vitro.
Three canine factors available in recombinant form (G-CSF,
GM-CSF, and SCF)44.45 were tested (Fig 5). The presence
of exogenous G-CSF or GM-CSF in MoAb-treated cultures
had no recognizable effect. However, SCF at 10 ng/mL or
higher completely prevented a decrease in CFU-GM that
were cultured from nonadherent cells. In addition, the adherent layer of SCF-treated cultures was largely maintained and
showed a prominence of large fibroblast-like cells with a
tendency to form clusters with nonadherent cells.
Anti-MHC Class II MoAb Treatment and Transmembrane
Signaling
Anti-MHC class I1 MoAbs, somewhat dependent upon
the recognized epitope, mediate transmembrane signals and
trigger Ca2’ mobilization in lymph~cytes.’~.~~
We hypothesized that similar events occur in canine marrow cells.
BM mononuclear cells were incubated with intact antiMHC class I1 MoAb (H81.9, BIF6, CA1.41, HBlOa, p4.1)
alone or in combination or with F(ab’), fragments of H81.9.
After 2 and 5 minutes, samples were procured and assayed
for tyrosine phosphorylation. No new protein bands and no
significant changes in the intensity of preexisting proteins
were detectable (Fig 6).
Next, marrow mononuclear cells, loaded with indo-l,
were treated with biotin-conjugated anti-MHC class I1
MoAb. There was no detectable change in [Ca*’]i with the
MoAb alone; a moderate signal was observed after crosslinking of biotin-conjugated H81.9 with avidin (Fig 7). Thus,
these results showed calcium signaling, but suggested that a
pathway other than tyrosine phosphorylation was involved.
Apoptosis in LTMCs
Nonadherent and adherentcellsfrom
LTMCs were
stained with acridine orange/ethidium bromideor alternatively subjected to the TUNEL method at various time
points after addition of MoAb H81.9 to cultures. Results
are illustrated inFig 8. As expected, some apoptoticnuclei
were seen even in control cultures. However, the proportion of cells showing apoptosis was increased in MoAbtreated LTMCs, both in the adherent and the nonadherent
MHCCLASSII-MEDIATEDINHIBITION
A
OF
HEMATOPOIESIS
Viability in non-adherent cells
3347
B
Viability in adherent cells
-"
"
"
..
OJ
.l
""
"
I
I
Dl
D2
D4
D7
D14
W
Fig 8. TUNEL staining ofMoAb-induced apoptosis in nonadherent
and adherent cells of LTMCs. MoAb H81.9 (10 pg/mL) wasadded t o
LTMCs at the time of recharging. On days 1, 2, 4, 7, and 14, cells
were examined for apoptosis. Shown separately for nonadherent (A)
and adherent cells IBI are the fractionof normal appearing (nonapop
totic) cells in control cultures (m),cultures treatedwith MoAb H81.9
(+l, and cultures treated with MoAb H81.9 plus SCF ( 9 . The results
on day 7 are shown in C through J: C, nonadherent cells, control; D,
adherent cells, control; E, nonadherent cells, SCF;F, adherent cells,
SCF; G, nonadherent cells, H81.9; H, adherent cells, H81.9; 1, nonadherent cells, H81.9 plus SCF; J, adherent cells, H81.9 plus SCF.
populations. As shown in Fig 8, the addition of exogenous
SCF to cultures significantly reduced the proportion of
apoptotic cells in H81.9-treated cultures. Overall, slightly
more cells survived in the nonadherent compartment than
among adherent cells.
Nonadherent and adherent cells from LTMCs were also
analyzed for DNA content byflow cytometry(Fig 9).
Beginning at 3 to 4 hours, apoptotic cell nuclei were de-
tectable. Apoptotic nuclei were seen among both adherent
and nonadherent cells. The proportions varied slightly
from experiment to experiment (ranging from 6% to 15%)
and reached a plateau at 24 hours. The concurrent presence
of exogenous SCF consistently reduced or prevented the
development of apoptosis. As shown in Fig IO, the electronmicroscopic appearance of treated cells was typical
for apoptosis.
HONG ET AL
3348
A
Control
H81.9 + SCF
H81.9
4 hrs
:L,
J
140
700
525
350
175
0
.........
0
256
M 192 128
0
DNA Content
B
Control
H81.9
H81.9 + SCF
24 hrs
0
64192 128
DNA Content
DISCUSSION
Cellular components of both the immune system and the
hematopoietic system are thought to be derived from the
same stem
and MHC genes are expressed in both
lymphoid and hematopoietic lineage^.'.^.*.^' However, although the role of MHC molecules within the immune system is well defined, their functions on hematopoietic cells
are less well understood. In particular, the function of class
I1 molecules on hematopoietic cells has remained controversial?'"' Expression is locus specific, lineage dependent, and
developmentally r e g ~ l a t e d . ' . ' . ~ . ~We
~ ~ ~have
'
previously
shown that MHC class I1 antigens are expressed on canine
hematopoietic cells that are required for sustained recovery
Fig 9. Increased apoptotic
DNA in cells from H81.9-treated
marrow cultures and prevention
of apoptosis by exogenous SCF.
Nonadherent (AIand adherent
cells (B) were analyzed at 4 and
24 hours after theaddition of
H81.9 (middle panel) or H81.9
plus SCF (right panel). Results
with cells fromuntreated cultures are shown in the left panel.
The vertical axis indicates cell
numbers, the horizontalaxis cellular DNA content. The dotted
line in each panel delineates the
original data. The continuing
lines delineate the cell cycle
phases calculated by the Multicycle program; the apoptotic peak
is shaded and the percentage of
apoptotic nuclei indicated by the
number in each panel.
after autologous marrow transplantation.2'.22Recently, we
observed that dogs given marrow-ablative doses of total
body irradiation (920 cGy) and infused with unmanipulated
autologous marrow fail to recover normal hematopoiesis and
die with marrow aplasia if treated with anti-MHC class I1
MoAb in the immediate posttransplant period (days 0
through 4)." To further investigate these observations, the
present in vitro studies were undertaken.
The addition of anti-MHC class I1 MoAb to LTMCs resulted in a dose-dependent decrease of CFU-GM precursors
among nonadherent cells. This was of note because no significant effect of these MoAbs was observed when added
directly to CFU-GM cultures in agar. Although it is possible
that, in a semisolid medium, MoAb would not be freely
MHC CLASS II-MEDIATED INHIBITION
h
i
3349
OF HEMATOPOIESIS
. ...
I
Fig 10. Ultrastructuralappearance of MoAb H81.9-treated nonadherent and adherent cells. Cells were cultured for 7 days in thepresence of MoAb H81.9. (A) nonadherent cells, (B) adherent cells. (Original magnification x 6,000.)
available and CFU-GM would not be exposed to concentrations sufficient for inhibition, this is unlikely because even
MoAb concentrations 20-fold higher than used in LTMCs
did not result in direct CFU-GM inhibition, and preincubation of CFU-GM with MoAb failed to result in an inhibitory
effect. Thus, these results suggest that the anti-MHC class
I1 MoAb exerted its effect not directly on CFU-GM, but
rather on a less mature precursor cell or, alternatively, on
nonhematopoietic accessory cells. These cells would be expected to express class I1 molecules. Indeed, morphologic
analyses in the present study showed that anti-MHC class I1
MoAb interfered with the development of a normal functional adherent layer, a finding in agreement with observations byBriihl et al who had shown that the presence of
anti-HLA-DR and -DP MoAb prevented the formation of
confluent adherent layers in human in vitro models." Those
results were surprising because MHC class 11 antigens are
generally thought to be expressed minimally or not at all?'.s3
They are, nevertheless, consistent withthefindingthatat
least subpopulations of canine marrow-derived stromal cells
express class
Those cells could support hematopoiesis
directly, eg, by serving as anchor or by providing growth
factors, or indirectly via the generation of signals necessary
for other supportive cells.
Consistent withthenotionthat
subpopulations of cells
are affected by anti-MHC class I1 MoAbis the analysis
of apoptosis, showing that only a proportion of cells, both
adherent and nonadherent, was involved. Apoptosis has been
observed in B lymphocytes exposed to MoAbs directed at
MHC class I1 and could beprevented by interleukin-4."
Binding of MoAb to class I1 antigens has also been shown
to induce tyrosine phosphorylation and C& flux in T lymphocytes." Superantigen-induced apoptosis of class I' T
cells is at least partially mediated by
early class I1 signals
from tyrosine kinase activation, but is also dependent upon
late adhesion through beta-2 integrins."." Experiments on
transmembrane signaling in the present study showed evidence for class 11-mediated calcium signals, although no
change in tyrosine phosphorylation was seen in whole cell
lysates. The role of adhesion, clearly MHC class I1 inducible
in B cell^^'^^^ remains to be determined in the present model.
Regardless of the pathway, MoAb-mediated apoptosis and
inhibition of hematopoiesis wereprevented by exogenous
SCF. Williams et al first reported that growth factor withdrawal from hematopoietic cultures in vitro resulted in
apoptosisy and others showed that apoptosis in human erythroid progenitors was prevented or reduced by SCF even
in the absence oferythropoietin."."However,thereisno
direct evidence that both MHC class 11 and c-kit-dependent
signals are mediated through the same cell. In fact, semiquantitative dilution experiments with highly enriched human CD34' cells assayed for colony formation in the absence or presence of MoAb H8 1.9suggest that an accessory
cell is involved in the MHC-mediated effect."' Such a hypothesis is also consistent with observations in the present
study: anti-MHC class I1 MoAb-triggered apoptotic cell
death was present in adherent and nonadherent cells; however, the addition of SCF maintained viability predominantly
among nonadherent cells. This could be readily explained
byan effect of MoAbs on accessory cells in the adherent
compartment, which serve as a source of SCF. Loss of those
cells, in turn, would result in cell death among nonadherent
cells. The addition of exogenous SCF would primarily affect
SCF-dependent nonadherent cells and much less so the adherent layer, although the SCF receptor c-kit appears to be
expressed on certain stromal cells."' Such a scenario would
also accommodate in vivo observations": while the administration of SCF abrogated marrow failure in H81.9-treated
dogs, a secondary decrease in granulocytes after the transient
initial recoverywas not completely prevented. These kinetics
are consistent with recovery from a small pool of surviving
stem cells rather than complete prevention of H81.9-induced
damage.
Interestingly, the addition of G-CSF or GM-CSF to
LTMCs failed to show anyeffect on stromal cells or hematopoiesis in the presence of anti-MHC class I1 MoAbs, suggesting that to overcome inhibition of hematopoiesis by antiMHC class 11 MoAb, an early acting viability maintaining
3350
HONG ET AL
factor such as SCF is required. If MoAb-mediated inhibition
of hematopoiesis involves downregulation of growth factors,
G-CSF and GM-CSF are unlikely candidates. In fact, preliminary data described elsewhere are consistent with this interpretation insofar as, at least atthemRNA
level, G-CSF
is not affected by anti-class I1 treatment, and GM-CSF i s
upregulated." However, stromal cells are also a major source
of SCF,6s,66and, particularly under stress conditions, optimum support of hematopoiesis by the stromal layer is required. Neta et a16' have recently shown in mice that postirradiation hematopoietic recovery isimpaired by treatment with
anti-SCF antibody. Similarly, we have shown thatneutralization of SCF in LTMCs using a specific MoAb results in
exhaustion of hematopoiesis."
In conclusion, anti-MHC class 11-mediated inhibition of
hematopoiesis and abrogation by the c-kit ligand SCF involves a complex mechanism, Programmed cell death represents one component. Additional factors include stromal cell
dysfunction and dysregulation of growth factors.
ACKNOWLEDGMENT
We appreciate the support ofAmgen lnc, andthe stimulating
discussion with Ian McNiece and Rick Jacobson. We thank Bonnie
Larson and Harriet Childs for typing the manuscript.
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