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Selective expansion of a monocyte subset expressing the CD11c dendritic cell marker in the Yaa model of systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 9, September 2005, pp 2790–2798
DOI 10.1002/art.21365
© 2005, American College of Rheumatology
Selective Expansion of a Monocyte Subset Expressing the
CD11c Dendritic Cell Marker in the Yaa Model of
Systemic Lupus Erythematosus
Hirofumi Amano,1 Eri Amano,1 Marie-Laure Santiago-Raber,1 Thomas Moll,1
Eduardo Martinez-Soria,1 Liliane Fossati-Jimack,2 Masahiro Iwamoto,1 Stephen J. Rozzo,3
Brian L. Kotzin,3 and Shozo Izui1
monocyte subset compared with the inflammatory subset, and the former expressed CD11c, a marker of
dendritic cells. Neither monocytosis nor the change in
monocyte subpopulations, including CD11c expression,
was observed in Yaa-bearing C57BL/6 mice, in which
systemic lupus erythematosus (SLE) fails to develop.
Conclusion. Our results suggest that Yaaassociated monocytosis is not attributable to an intrinsic abnormality in the growth potential of monocyte
lineage cells bearing the Yaa mutation and that the
Yaa mutation could lead to the expansion of dendritic
cells, thereby contributing to the accelerated development of SLE.
Objective. Monocytosis is a unique cellular abnormality associated with the Yaa (Y-linked autoimmune
acceleration) mutation. The present study was designed
to define the cellular mechanism responsible for the
development of monocytosis and to characterize the
effect of the Yaa mutation on the development of monocyte subsets.
Methods. We produced bone marrow chimeras
reconstituted with a mixture of Yaa and non-Yaa bone
marrow cells bearing distinct Ly-17 alloantigens, and
determined whether monocytes of Yaa origin became
dominant. Moreover, we defined the 2 major inflammatory (Gr-1ⴙ,CD62 ligand [CD62L]ⴙ) and resident (Gr1ⴚ,CD62Lⴚ) subsets of blood monocytes in aged BXSB
Yaa male mice, as compared with BXSB male mice
lacking the Yaa mutation.
Results. Analysis of the Ly17 allotype of blood
monocytes in chimeric mice revealed that monocytes of
both Yaa and non-Yaa origin were similarly involved in
monocytosis. Significantly, the development of monocytosis paralleled a selective expansion of the resident
In the BXSB strain of mice, an autoimmune
syndrome with features of systemic lupus erythematosus
(SLE) develops spontaneously, and male mice are affected much earlier than female mice (1). The accelerated development of SLE in male BXSB mice is attributable to the presence of an as-yet-unidentified mutant
gene located on the Y chromosome, designated Yaa
(Y-linked autoimmune acceleration) (2). The Yaa gene
by itself is unable to induce significant autoimmune
responses in mice without an apparent SLE background,
but in combination with autosomal susceptibility alleles
that are present in lupus-prone mice, it can induce and
accelerate the development of SLE (3,4).
Monocytosis is a unique cellular abnormality
associated with the Yaa mutation (5). In peripheral
blood mononuclear cells (PBMCs) from 8-month-old
male BXSB Yaa mice, the frequency of monocytes
reaches ⬎50%. The development of monocytosis is
apparently dependent on the progression of SLE because monocytosis was not observed in BXSB.ll (ll for
Supported by the Swiss National Foundation for Scientific
Research and the NIH (grant AR-37070). Dr. Fossati-Jimack is
recipient of a fellowship from the Arthritis Research Campaign, UK.
1
Hirofumi Amano, MD, Eri Amano, MD, Marie-Laure
Santiago-Raber, PhD, Thomas Moll, PhD, Eduardo Martinez-Soria,
PhD, Masahiro Iwamoto, MD, Shozo Izui, MD: University of Geneva,
Geneva, Switzerland; 2Liliane Fossati-Jimack, PhD: Imperial College
School of Medicine, London, UK; 3Stephen J. Rozzo, PhD, Brian L.
Kotzin, MD: University of Colorado Health Sciences Center, Denver.
Drs. Hirofumi Amano and Eri Amano contributed equally to
this work.
Address correspondence and reprint requests to Shozo Izui,
MD, Department of Pathology and Immunology, Centre Médicale
Universitaire, 1211 Geneva 4, Switzerland. E-mail: Shozo.Izui@
medecine.unige.ch.
Submitted for publication March 23, 2005; accepted in revised
form May 31, 2005.
2790
Yaa GENE–LINKED MONOCYTOSIS
long-lived) and BXSB.H2d mice, both of which fail to
develop SLE (6,7). However, such a monocytosis is not
a common feature of 2 lupus-prone strains of mice
([NZB ⫻ NZW]F1 and MRL-Faslpr), indicating that this
abnormality is causally linked to the Yaa mutation.
Monocytes newly generated in the bone marrow
circulate for a few days before entering tissues, where
they differentiate into mature resident macrophages (8).
Under conditions of inflammation, monocyte production in the bone marrow is increased, and the cells are
rapidly recruited to sites of inflammation, where they
differentiate into inflammatory macrophages (9). Recent studies in mice demonstrated that circulating
monocytes could be divided into 2 phenotypically and
functionally distinct subsets (10,11). The first subset of
monocytes, which is classified as “inflammatory” and is
characterized by a Gr-1⫹,CX3CR1low,CCR2⫹,CD62 ligand (CD62L)⫹ phenotype, is preferentially recruited
to inflamed tissue. The second subset of monocytes,
Gr-1⫺,CX3CR1high, CCR2⫺,CD62L⫺, is considered to
be a source of tissue resident macrophages and dendritic
cells (DCs), and is classified as “resident.” These 2
subsets correspond, respectively, to the CCR2⫹,CD16⫺
and CCR2⫺,CD16⫹ monocyte populations described in
humans (12). Because blood monocytes are the major
source of DCs, it is important to determine the effect of
the Yaa mutation on development of the 2 different
subsets of monocytes in BXSB Yaa male mice in relation
to acceleration of the disease.
In the present study, we first analyzed whether the
development of monocytosis is a defect intrinsic to monocyte lineage cells bearing the Yaa mutation or is associated
with excessive production of a monocyte-specific growth
factors(s) in Yaa-bearing mice. To address this question,
we constructed radiation bone marrow chimeric mice
reconstituted with a mixture of Yaa and non-Yaa bone
marrow cells bearing distinct Ly17 alloantigens, and determined whether monocytes of Yaa origin became dominant.
Second, we defined the 2 major subsets of blood monocytes
in aged male BXSB Yaa mice as compared with male
BXSB mice lacking the Yaa mutation. Our results demonstrated that monocytes of both Yaa and non-Yaa origin
were similarly involved in the development of monocytosis.
In addition, a selective expansion of resident monocytes
expressing CD11c, a marker of DCs, suggests that the Yaa
mutation may lead to excessive production of DCs, thereby
contributing to acceleration of lupus autoimmune responses.
2791
MATERIALS AND METHODS
Mice. BXSB and NZB (Ly-17.1) mice were purchased
from The Jackson Laboratory (Bar Harbor, ME). Accelerated
SLE develops in male BXSB mice, but not in female BXSB
mice, partly because of the action of the Yaa gene (1). BXSB
mice lacking the Yaa gene, C57BL/6 (B6; Ly17.2) mice bearing
the Yaa mutation (B6.Yaa), and B6.NZB-Nba2 congenic mice
homozygous for the Nba2 (New Zealand black autoimmunity
2) lupus-susceptibility locus and bearing the Ly17.1 allele
(B6.Ly17.1) were generated as described previously (4,13,14).
B6.Ly17.1 mice bearing the Yaa mutation were then generated
by intercrossing B6.Ly17.1 females and B6.Yaa males. (NZB ⫻
B6.Ly17.1.Yaa)F1 (Ly17.1/Ly17.1) and (NZB ⫻ B6)F1 (Ly17.1/
Ly17.2) mice were bred in the animal facility of the Centre
Médical Universitaire, Geneva.
Flow cytometric analysis. Flow cytometry was performed using 2- or 3-color staining of peripheral blood mononuclear cells (PBMCs) and analyzed with a FACSCalibur
cytometer (BD Biosciences, San Jose, CA). The following
antibodies were used: anti-F4/80, anti-CD11b (M1/70), anti–
Ly-6C/G (Gr-1), anti-CD62L (MEL-14), anti–Ly-17.2
(K9.361), anti-CD11c (N418), anti–I-A (Y-3P), anti-CD80
(1G10), anti-CD86 (GL1), anti-CD4 (GK1.5), anti-CD8␣
(5H10-1), anti-B220 (RA3-6B2), anti-CD45RA (14.8), and
anti-CD19 (1D3) monoclonal antibodies.
Serologic analysis. Serum levels of IgG anti-DNA
autoantibodies were determined by enzyme-linked immunosorbent assay, with results expressed as titration units (units/
ml) in reference to a standard curve obtained from lupusprone MRL-Faslpr mice, as described previously (15).
In vivo monocyte proliferation assay. Mice received 6
mg of bromodeoxyuridine (BrdU; Sigma-Aldrich, St. Louis,
MO) intravenously. Two days later, peripheral blood monocytes were stained for CD11b, F4/80, and Gr-1 and then for
BrdU incorporation (BrdU Flow kit; BD Biosciences).
Preparation of double bone marrow chimeras. Threeto-four–month-old (NZB ⫻ B6)F1 recipient mice were irradiated (850 rads) and reconstituted with a mixture of bone
marrow cells (5 ⫻ 106 from each donor) from 3-to-4–monthold male Ly17.1/Ly17.1 (NZB ⫻ B6.Ly17.1.Yaa)F1 Yaa mice
and male Ly17.1/Ly17.2 (NZB ⫻ B6)F1 non-Yaa mice or, as a
control, a mixture of bone marrow cells from male Ly17.1/
Ly17.1 (NZB ⫻ B6.Ly17.1)F1 and Ly17.1/Ly17.2 (NZB ⫻
B6)F1 non-Yaa mice, as described previously (16). Two months
later, chimerism in recipients was assessed by staining peripheral blood B cells with anti–Ly-17.2 (K9.361) and anti-B220
(RA3-6B2) monoclonal antibodies.
Statistical analysis. Statistical analysis was performed
with Wilcoxon’s 2-sample test. P values less than or equal to
5% were considered significant.
RESULTS
Age-dependent development of monocytosis in
lupus-prone male (NZB ⴛ B6. Yaa)F1 and BXSB Yaa
mice, but not in male B6.Yaa mice. As described previously (5), 8-month-old male BXSB Yaa mice had an
2792
AMANO ET AL
Table 1. Development of monocytosis, anti-DNA autoantibodies,
and GN in lupus-prone male (NZB ⫻ B6.Yaa)F1 and BXSB mice
bearing the Yaa mutation*
Mice
Yaa
Monocytes
Anti-DNA
50% mortality
due to GN
BXSB
BXSB
(NZB ⫻ B6)F1
(NZB ⫻ B6)F1
B6
B6
⫹
–
⫹
–
⫹
–
46.9 ⫾ 8.9
14.4 ⫾ 4.6
39.0 ⫾ 8.1
14.7 ⫾ 3.1
14.0 ⫾ 4.4
10.3 ⫾ 3.0
67 ⫾ 36
21 ⫾ 9
81 ⫾ 50
20 ⫾ 8
11 ⫾ 8
3⫾2
8 months
⬎20 months
14 months
⬎20 months
⬎20 months
⬎20 months
* Values are the mean ⫾ SD of 8–20 mice in each group. The
percentage of CD11b⫹,F4/80⫹ monocytosis in peripheral blood mononuclear cells at 8 months of age was determined by flow cytometry. At
2 months of age, the percentage of blood monocytes in different
groups of mice tested in the present study was ⬃10% (data not shown).
Serum levels of IgG anti-DNA, expressed as units/ml, at 8 months of
age were determined by enzyme-linked immunosorbent assay. GN ⫽
glomerulonephritis.
increased percentage of monocytes in blood, while this
age-dependent monocytosis was not observed in male
BXSB non-Yaa mice, which are unable to develop a
lupus-like autoimmune syndrome (13) (Table 1). Because the Yaa mutation induces severe SLE in male
(NZB ⫻ B6.Yaa)F1 mice, with 50% mortality at 14
months of age (17), but fails to induce significant
lupus-like autoimmune disease in nonautoimmune B6
mice (4), the development of monocytosis was assessed
in these mice. Eight-month-old male (NZB ⫻ B6.Yaa)F1
mice displayed a significant monocytosis, with 26–52%
of PBMCs being monocytes (P ⬍ 0.001) (Table 1).
Monocytosis did not develop in male (NZB ⫻ B6)F1
mice lacking the Yaa mutation, and these mice also
failed to develop disease. Notably, the absolute number
of monocytes increased in male (NZB ⫻ B6.Yaa)F1 mice
in which monocytosis developed (data not shown), as
was observed in male BXSB Yaa mice (5). In contrast,
CD11b⫹,F4/80⫹ monocytes in 8-month-old male
B6.Yaa mice represented not more than 20% of PBMCs,
and this percentage was comparable with that in control
male B6 mice. These results thus confirmed a close
association of monocytosis with the Yaa-mediated lupuslike autoimmune disease.
Increased production of blood monocytes in aged
male BXSB Yaa mice. To investigate whether the development of monocytosis in aged male BXSB Yaa mice
was indeed the result of increased generation of monocytes in the bone marrow rather than the result of
abnormal persistence in the circulating blood, 8-monthold male BXSB mice bearing or lacking the Yaa mutation were treated intravenously with BrdU. Flow cyto-
Figure 1. Comparable expansion of the populations of monocytes of
both Yaa and non-Yaa origin in radiation bone marrow chimeras.
Irradiated (NZB ⫻ B6)F1 mice were reconstituted with a mixture of
bone marrow cells from male Ly17.1/Ly17.1 (NZB ⫻ B6.Ly17.1.Yaa)F1
Yaa mice and male Ly17.1/Ly17.2 (NZB ⫻ B6)F1 non-Yaa mice or, as
a control, a mixture of bone marrow cells from male (NZB ⫻
B6.Ly17.1)F1 and (NZB ⫻ B6)F1 non-Yaa mice. A, Two months after
reconstitution, peripheral blood mononuclear cells (PBMCs) were
stained with a combination of anti–Ly-17.2 and anti-B220 monoclonal
antibodies. Representative staining profiles for the Ly17 allotype of
B220⫹ B cells from Yaa plus non-Yaa chimeras and control chimeras
are shown. Numbers in the boxes are the percentages of Ly-17.2⫺ (Yaa
origin) and Ly-17.2⫹ (non-Yaa origin) B220⫹ B cells. B, Eight months
after reconstitution, PBMCs were stained with a combination of
anti-F4/80, anti–Ly-17.2, and anti-B220 monoclonal antibodies. Representative staining profiles for F4/80⫹ and B220⫹ cells and for the
Ly17 allotype of F4/80⫹ monocytes from Yaa plus non-Yaa chimeras
and control chimeras are shown. Numbers in the boxes are the
percentages of F4/80⫹ monocytes, and of Ly-17.2⫺ (Yaa origin) and
Ly-17.2⫹ (non-Yaa origin) F4/80⫹ monocytes.
Yaa GENE–LINKED MONOCYTOSIS
metric analysis of blood monocytes 2 days after BrdU
injection revealed that the percentages of
BrdU⫹,CD11b⫹ monocytes in PBMCs from male
BXSB Yaa mice (mean ⫾ SD 6.7 ⫾ 1.7%) were ⬃5-fold
higher than those in PBMCs from male non-Yaa BXSB
mice (mean ⫾ SD 1.3 ⫾ 0.4%; P ⬍ 0.001). Notably, the
percentage of BrdU⫹ cells among CD11b⫹ monocytes
was comparable in male BXSB Yaa mice (mean ⫾ SD
10.6 ⫾ 1.9%) and their non-Yaa counterparts (10.3 ⫾
3.8%). These results indicate that monocytosis observed
in aged male BXSB Yaa mice is caused by an increased
production of monocytes in bone marrow and their
subsequent release into the circulation, and not by an
aberrant accumulation of monocytes in the circulating
blood due to differences in migration into tissues.
Comparable involvement of monocytes of both
Yaa and non-Yaa origin in the development of monocytosis. The increased production of monocytes in male
BXSB Yaa mice could be attributable either to excessive
production of monocyte-specific growth factor(s) or to
hyperresponsiveness of monocyte lineage cells to normal
levels of growth factors. To address this question, irradiated (NZB ⫻ B6)F1 mice were reconstituted with a
mixture of bone marrow cells from Ly17.1 homozygous
male (NZB ⫻ B6.Ly17.1.Yaa)F1 Yaa mice and Ly17.1/
Ly17.2 heterozygous male (NZB ⫻ B6)F1 non-Yaa mice,
in which Ly17.1 and Ly17.2 are allelic markers of CD32.
The enumeration of Ly-17.2⫹ circulating B cells (defined by B220 staining) 2 months after reconstitution
showed that the percentage of Ly-17.2⫹ B cells (mean ⫾
SD 22.7 ⫾ 3.0% [n ⫽ 12 mice]) was comparable with the
percentage of Ly-17.2⫺ B cells (25.8 ⫾ 4.1%), confirming an equal reconstitution of hematopoietic cells derived from both Yaa and non-Yaa origin (Figure 1A).
Notably, similar results were obtained with control chimeras, in which irradiated F1 mice were reconstituted
with a mixture of bone marrow cells from male (NZB ⫻
B6.Ly17.1)F1 and (NZB ⫻ B6)F1 mice, both of which
lack the Yaa mutation (for Ly17.2⫹ B cells, mean ⫾ SD
24.3 ⫾ 5.4%; for Ly17.2⫺ B cells, 26.2 ⫾ 4.1% [n ⫽ 7
mice]).
In Yaa plus non-Yaa (B6 ⫻ NZB)F1 bone marrow chimeras, increased percentages of monocytes developed 8 months after reconstitution (mean ⫾ SD
29.0 ⫾ 7.2% [n ⫽ 12 mice]) as compared with control
non-Yaa chimeras (mean ⫾ SD 10.3 ⫾ 2.9% [n ⫽ 7
mice]; P ⬍ 0.001) (Figure 1B). Analysis of the Ly-17
alloantigen on F4/80⫹ blood monocytes revealed that
the percentage of Ly17.2⫺ monocytes of Yaa origin
(mean ⫾ SD 15.7 ⫾ 4.9%) was comparable with the
2793
Figure 2. Selective expansion of the Gr-1⫺,CD62 ligand (CD62L)⫺
monocyte subset in aged male BXSB Yaa mice developing monocytosis. Peripheral blood mononuclear cells from 2- and 8-month-old male
BXSB and B6 mice of the Yaa or non-Yaa genotype were stained with
a combination of anti-CD11b, anti–Gr-1, and anti-CD62L monoclonal
antibodies. A, Representative staining profiles for Gr-1 in relation to
staining with anti-CD11b monoclonal antibodies. Numbers in the
boxes are the percentages of Gr-1⫹ and Gr-1⫺,CD11b⫹ monocytes.
B, Histograms showing CD62L staining in the Gr-1⫹ and Gr-1⫺
monocyte subsets from 2- and 8-month-old male BXSB Yaa mice. The
results obtained for differential CD62L staining in the 2 monocyte
subsets were identical in aged male BXSB non-Yaa and B6.Yaa mice
(data not shown).
2794
AMANO ET AL
Table 2. Selective expansion of Gr-1⫺,CD11b⫹ monocytes in aged
male BXSB Yaa, but not B6.Yaa, mice*
Mice
Yaa
Age
Gr-1⫹
Gr-1⫺
BXSB
BXSB
BXSB
BXSB
B6
B6
B6
B6
⫹
–
⫹
–
⫹
–
⫹
–
2 months
2 months
8 months
8 months
2 months
2 months
8 months
8 months
5.5 ⫾ 1.0
3.6 ⫾ 0.9
4.7 ⫾ 1.7
3.9 ⫾ 0.7
3.9 ⫾ 1.1
3.0 ⫾ 1.0
5.0 ⫾ 0.7
3.3 ⫾ 0.7
5.5 ⫾ 2.2
4.1 ⫾ 0.9
29.5 ⫾ 9.1
6.0 ⫾ 3.0
3.6 ⫾ 1.4
3.8 ⫾ 1.7
4.9 ⫾ 2.7
4.6 ⫾ 2.2
* Percentage of Gr-1⫹ and Gr-1–,CD11b⫹ monocytes in peripheral
blood mononuclear cells from male BXSB and B6 mice (mean ⫾ SD
of 5–10 mice from each group).
percentage of Ly-17.2⫹ monocytes of non-Yaa origin
(14.0 ⫾ 26%) (Figure 1B). Notably, in control chimeras
reconstituted with a mixture of non-Yaa Ly17.1 plus
Ly17.1/Ly17.2 donor cells, the respective monocyte populations in circulating blood were of equal size (for
Ly17.2⫹ monocytes, mean ⫾ SD 4.4 ⫾ 1.3%; for
Ly17.2⫺ monocytes, 5.4 ⫾ 2.1%). These results indicate
no selective production of monocytes from the Yaabearing bone marrow cells, thus providing evidence
against selective hyperresponsiveness to growth factors
of Yaa-bearing monocyte lineage cells.
Selective expansion of the resident monocyte
subset in aged male BXSB Yaa mice but not in male B6
Yaa mice. Recent studies have demonstrated the existence of 2 major subpopulations of blood monocytes,
resident and inflammatory monocytes, which apparently
represent 2 phenotypically and functionally distinct subsets (10,11). To determine whether the Yaa mutation
could exhibit an effect on the development of these 2
subsets, the expression of Gr-1 on blood monocytes from
male BXSB Yaa and non-Yaa mice was analyzed by flow
cytometry. In 2-month-old BXSB mice, independent of
the Yaa genotype, the percentage of cells expressing the
Gr-1⫹,CD11b⫹ monocyte phenotype in the circulation
was almost the same as the percentage of cells expressing the Gr-1⫺,CD11b⫹ monocyte subset (Figure 2A
Figure 3. Dominance of the Gr-1⫹ monocyte subset in bromodeoxyuridine (BrdU)⫹ recent immigrant cells
from bone marrow in male BXSB Yaa and non-Yaa mice. Two days after a single injection of BrdU into
8-month-old male BXSB Yaa and non-Yaa mice, peripheral blood mononuclear cells were stained with a
combination of anti-F4/80, anti-BrdU, and anti–Gr-1 monoclonal antibodies and gated for F4/80⫹ cells.
Representative results from each group of mice are shown. Numbers in the boxes are the percentages of each
population stained with anti-BrdU and anti–Gr-1 monoclonal antibodies. For Yaa mice (n ⫽ 7), the mean ⫾ SD
percentage of each population is as follows: for Gr-1⫹, BrdU⫹, 7.6 ⫾ 2.2%; for Gr-1⫺, BrdU⫹, 1.7 ⫾ 0.6%;
for Gr-1⫹, BrdU⫺, 12.3 ⫾ 5.4%; and for Gr-1⫺, BrdU⫺, 78.9 ⫾ 6.5%. For non-Yaa mice (n ⫽ 5), the mean ⫾
SD percentage of each population is as follows: for Gr-1⫹, BrdU⫹, 7.4 ⫾ 0.8%; for Gr-1⫺, BrdU⫹, 1.5 ⫾
1.2%; for Gr-1⫹, BrdU⫺, 44.3 ⫾ 3.1%; and for Gr-1⫺, BrdU⫺, 47.0 ⫾ 2.5%.
Yaa GENE–LINKED MONOCYTOSIS
and Table 2). Similar percentages of GR-1⫹ and Gr-1⫺
monocytes were present in nonautoimmune B6 mice.
However, in 8-month-old male BXSB Yaa mice developing
monocytosis, the Gr-1⫺ monocyte subset selectively increased (⬃6-fold more than the Gr-1⫹ subset; P ⬍ 0.001)
and became the dominant monocyte population in the
circulating blood. Notably, this population also lacked
expression of CD62L (Figure 2B), indicating that it belonged to the resident monocyte subset, while Gr-1⫹
monocytes were CD62L⫹, corresponding to the inflammatory monocyte subset, as described by Geissmann et al
(10). In contrast, the balance of these 2 subsets did not
change in aged male BXSB mice lacking the Yaa mutation
or in aged male B6.Yaa mice (Figure 2A).
It was unclear whether these 2 monocyte subsets
are derived from different monocyte lineages or whether
they originate from the same precursor, with one differentiating into the other in the circulating blood. To
address this question, 2 days after an intravenous injection of BrdU in 8-month-old male BXSB Yaa mice,
surface expression of Gr-1 on newly generated
BrdU⫹,F4/80⫹ monocytes was examined by flow cytometry. The majority of BrdU⫹,F4/80⫹ recent immigrant
cells from the bone marrow stained positively with Gr-1
in both male BXSB Yaa and non-Yaa mice, independent
of the proportion of Gr-1 phenotypes among preexisting
unlabeled monocytes (Figure 3). This strongly suggests
that Gr-1⫹ monocytes likely became the Gr-1⫺ subset
while still in the blood, thus providing evidence against
the presence of 2 different monocyte lineages at the level
of bone marrow.
Increased expression of CD11c on the resident
monocyte subset in aged male BXSB Yaa, but not B6
Yaa, mice. To further characterize the 2 different subpopulations of monocytes present in aged male BXSB
Yaa mice, we determined the expression of different
surface markers. As described previously (5,10), both
populations of monocytes expressed neither class II
major histocompatibility complex (MHC) molecules nor
costimulatory CD80 and CD86 (data not shown). However, the Gr-1⫺ subset of monocytes from 8-month-old
male BXSB Yaa mice displayed significant surface expression of CD11c, as compared with Gr-1⫹ monocytes,
which remained negative for CD11c (Figure 4). This
enhanced expression was not observed in Gr-1⫺ monocytes from 2-month-old male BXSB Yaa mice. Gr-1⫺
monocytes in 8-month-old male BXSB non-Yaa and
B6.Yaa mice minimally expressed CD11c, at levels much
lower than those seen in aged male BXSB Yaa mice (P ⬍
0.001) (Figure 4). The age-dependent, selective increase
2795
Figure 4. Increased CD11c expression on the Gr-1⫺ monocyte subset
from aged BXSB Yaa mice developing monocytosis. Peripheral blood
mononuclear cells from 2- and 8-month-old male BXSB and B6 mice
of the Yaa or non-Yaa genotype were stained with a combination of
anti-CD11b, anti–Gr-1, and anti-CD11c monoclonal antibodies and
gated for CD11b⫹ cells. Representative staining profiles for CD11c in
the Gr-1⫹ and Gr-1⫺ monocyte subsets are shown. Values are the
mean ⫾ SD fluorescence intensity of CD11c (5–7 mice per group).
of Gr-1⫺,CD11c⫹ monocytes was similarly observed in
male (NZB ⫻ B6.Yaa)F1 mice in which SLE developed
(data not shown). Notably, these Gr-1⫺,CD11c⫹ monocytes in aged male BXSB and (NZB ⫻ B6.Yaa) F1 Yaa
mice did not express CD4, CD8␣, and B220 at a
detectable level (data not shown).
Because a recent study identified a CD11clow,
CD11b⫺,CD45RA⫹,B220⫹ population of cells, resembling precursors of plasmacytoid DCs, in the peripheral
blood of mice (18), we explored the possible expansion
of this particular subset in aged male BXSB Yaa mice.
However, CD11b⫺,CD45RA⫹,B220⫹ cells were barely
detectable among CD19⫺ non–B cells in circulating
blood from these mice (data not shown).
2796
AMANO ET AL
DISCUSSION
Monocytosis is a unique cellular abnormality
associated with Yaa-mediated lupus-like autoimmune
disease (5). The present study was designed to define the
cellular mechanism responsible for the development of
monocytosis and to characterize the effect of the Yaa
mutation on the development of 2 different subsets of
monocytes. We provide evidence that in mixed bone
marrow chimeras, monocytes of both Yaa and non-Yaa
origin were similarly involved in the development of
monocytosis, suggesting that monocytosis is not attributable to an intrinsic abnormality in the growth potential
of monocyte lineage cells in mice bearing the Yaa
mutation. Furthermore, we observed that monocytosis
resulted in selective expansion of a Gr-1⫺,CD62L⫺
monocyte subset expressing the CD11c DC marker.
Thus, the Yaa mutation could lead to the expansion of
cells that develop into DCs in the tissues, thereby
contributing to the acceleration of autoimmune responses in lupus-prone mice.
Analysis of Yaa plus non-Yaa mixed bone marrow
chimeras clearly demonstrated that there was no selective production of monocytes of Yaa origin over those of
non-Yaa origin. This result strongly suggests that monocytosis associated with the Yaa mutation is not attributable to hyperresponsiveness of monocyte lineage cells
bearing the Yaa mutation to normal levels of monocytespecific growth factor(s), but more likely, is a result of
excessive production of growth factors during the course
of lupus-like autoimmune syndrome. Because Yaalinked monocytosis is associated with the development
of SLE, one attractive hypothesis is that the activation of
macrophages, for example by autoantigen-antibody immune complexes produced during the course of the
disease, leads to an excessive production of monocytespecific growth factor(s) by macrophages, resulting in
the development of monocytosis in BXSB Yaa mice. It
has been shown that the interaction of immune complexes with IgG Fc␥ receptor (Fc␥R) on macrophages
triggers the production of monocyte-specific growth
factors, such as monocyte colony-stimulating factor and
granulocyte–macrophage colony-stimulating factor
(GM-CSF) (19,20). Because lupus-prone mice that are
deficient in activating Fc␥R spontaneously develop autoantibodies at levels comparable with those in wild-type
animals (21,22), it would be of interest to determine
whether BXSB mice deficient in Fc␥R are still able to
develop monocytosis, given the active production of
lupus autoantibodies.
The second major observation in the present
study is that the monocytosis that occurs in lupus-prone
Yaa mice is associated with a selective expansion of only
1 of the 2 major monocyte subsets present in the
circulation. This Gr-1⫺,CD62L⫺ subset has been considered a source of resident macrophages and DCs in
different tissues (10). Selective expansion of the Gr1⫺,CD62L⫺ monocyte subset in aged male BXSB Yaa
mice is consistent with the earlier finding that these mice
displayed hyperplasia of Kupffer cells (23), which are
considered to be derived from the Gr-1⫺ resident
monocyte subset (10).
The analysis of BrdU labeling of monocytes in
BXSB Yaa mice indicated that recent immigrants from
bone marrow enter the circulation as Gr-1⫹ monocytes.
This observation is consistent with the recent demonstration that monocytes repopulating the circulation
after depletion of blood monocytes by liposome treatment were exclusively of the Gr-1⫹ subset (11). In
addition, in vitro studies have demonstrated progressive
down-regulation of Gr-1 during culture of blood monocytes (24). Thus, it is probable that Gr-1⫹ and Gr-1⫺
monocytes represent 2 different stages of maturation,
during which the Gr-1⫹ subset becomes the more
mature Gr-1⫺ subset while still in the bloodstream.
Based on the differential expression of chemokine receptors and adhesion molecules by these 2 subsets, it has
been suggested that Gr-1⫹ monocytes could more efficiently migrate into inflamed tissues (10). Because the
production of monocytes in the bone marrow is stimulated during peripheral inflammation, it may be beneficial for the host to have high numbers of immature
Gr-1⫹ monocytes, which have a high potential for
migration into sites of inflammation (10,25), released
from the bone marrow.
Importantly, we observed that the Gr-1⫺ subset
expressed substantial levels of CD11c in aged lupusprone male BXSB and (NZB ⫻ B6.Yaa)F1 Yaa mice. In
contrast, expression of CD11c was minimal or absent on
the same subset in nonautoimmune mice as well as in
young BXSB and (NZB ⫻ B6.Yaa)F1 Yaa mice. The
molecular mechanisms responsible for the expansion of
Gr-1⫺ monocytes and for the induction of CD11c
expression remain to be determined. One hypothesis is
that the presence of the Yaa mutation in lupus-prone
mice may induce a unique cytokine environment, possibly through the activation of Fc␥R by immune complexes, as discussed above, not only promoting the rapid
maturation toward the Gr-1⫺ subset, but also inducing
an increased level of CD11c expression. Although it has
Yaa GENE–LINKED MONOCYTOSIS
been established that CD11c is a marker of DCs, the
expanding population of monocytes in aged male BXSB
Yaa mice express neither class II MHC molecules nor
costimulatory CD80 and CD86. It is possible that these
cells could be the precursors of DCs.
A recent study identified 2 major populations
of DC precursors in mouse blood, CD11cintermediate,
CD11b⫹,CD45RA⫺,B220⫺ and CD11clow,CD11b⫺,
CD45RA⫹,B220⫹ (18). The surface phenotype of
CD11c⫹ monocytes that is abundantly present in BXSB
Yaa mice is comparable with that of the former cell type,
which can give rise to myeloid DCs after incubation with
GM-CSF and tumor necrosis factor ␣ (18). Thus, one
can speculate that the presence of an increased number
of Gr-1⫺,CD11c⫹ monocytes may provide a source of
resident DCs in the tissues, thereby accelerating autoimmune responses in male BXSB Yaa mice. It should be
stressed that aged BXSB and (NZB ⫻ B6.Yaa)F1 Yaa
mice did not display an apparent expansion in blood of
the second population of CD11b⫺ DC precursors resembling precursors of plasmacytoid DCs, for which a
role in the development of SLE has recently been
proposed (26).
Based on the selective production of anti-DNA
autoantibodies by B cells bearing the Yaa mutation in
Yaa plus non-Yaa mixed bone marrow chimeras (16,27),
we previously proposed that the Yaa defect may decrease the threshold of B cell receptor–mediated signaling, thereby triggering and excessively stimulating autoreactive B cells (2). This is consistent with our recent
finding that the Yaa mutation triggers the activation of
autoreactive B cells in a T cell–independent manner
early in life (28). In addition to this thesis, our present
results suggest that Yaa could accelerate the progression
of SLE through increased production of monocytes and
DCs. Clearly, further understanding of the mechanisms
responsible for Yaa-induced B cell activation and monocytosis should help identify the molecular nature of the
Yaa mutation and the pathogenesis of SLE.
ACKNOWLEDGMENTS
We thank Mr. G. Brighouse and Mr. G. Celetta for
their excellent technical assistance.
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