| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Institut National de la Santé et de la Recherche Médicale Unité 25,
Unité 345, and
Centre National de la Recherche Scientifique Unité Mixte de Recherche 8603, Hôpital Necker, Paris, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-10 were modified. Despite the presence of immune
complexes in their kidney glomeruli, no inflammation ensued, and serum
IL-12 and soluble TNF receptors remained at pre-disease levels. This
uncoupling of immune complex deposition and kidney damage resulted from
a local down-modulation of Fc
RIII (CD16) expression within the
glomeruli by G-CSF. Our results demonstrate a beneficial effect of high
doses of G-CSF in the prevention of lupus nephritis that may hold
promise for future clinical applications, provided caution is taken in
dose adjustment. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
G-CSF treatment likewise modifies the monocyte/macrophage lineage, both quantitatively and functionally, as assessed, respectively, by increased cell counts in the blood and a shift to an antiinflammatory pattern (3) that conferred protection in murine models of experimental endotoxemia (4).
The T cell compartment is also affected because G-CSF-mobilized blood cells display a significant reduction in T cell responses (5). Alloreactive responses are decreased by treatment with G-CSF as assessed by the reduced acute rejection of liver transplants (6) and the lower incidence of graft-versus-host disease (GVHD)2 in recipients of G-CSF-mobilized progenitor cells (7).
Other studies in mice have confirmed that treatment with G-CSF confers a significant protection against acute GVHD (8) that correlates with a down-regulation of Th1-derived cytokines and an increased production of Th2-derived cytokines. These T cell effects of G-CSF are most probably mediated indirectly, because receptors for G-CSF have not been detected on this cell type (1).
In autoimmune diseases, and particularly in systemic lupus
erythematosus, polarized T cell phenotypes (Th1 vs Th2) often display a
complex relationship to the pathogenesis. Systemic lupus erythematosus
is a humoral autoimmune syndrome, characterized by the serological
appearance of antinuclear autoantibodies. Glomerular immune complex
deposition may lead to renal failure and mortality.
MRL-lpr/lpr mice spontaneously develop a severe form of
lupus-like disease. Recent studies (9) have demonstrated
that disease acceleration correlated with increased IgG2a/IgG1 and
IgG3/IgG1 isotype ratios of the autoantibodies as well as increased
production of IFN- over IL-4 by spleen and lymph node CD4 T cells.
Several reports have demonstrated that neutralization of Th1 cytokines
was beneficial in lupus treatment. Indeed, MRL-lpr/lpr mice
deficient for the IFN- gene (10, 11) or the IFN-
receptor gene (12) were protected from disease, as were
(NZB x NZW)F1 mice treated with
anti-IFN- Abs (13) or IFN--soluble receptors
(14). Similarly, MRL-lpr/lpr mice treated with
anti-IL-12 Abs showed enhanced survival (15).
Moreover, (NZW x C57BL/6.Yaa)F1
mice transgenic for IL-4 (16) exhibited an antigenic
response shifted toward the Th2 phenotype and did not develop the
disease. However, Th2-derived cytokines may also play a deleterious
role, because MRL-lpr/lpr mice deficient for the IL-4 gene
were protected from disease (10), and anti-IL-4 Ab
prevented the onset of lupus nephritis in (NZB x
NZW)F1 mice (17).
In addition to this complex interplay of Th1/Th2 cytokines at the
initial phase of the disease, an inflammatory reaction develops at a
more advanced stage, and IL-12 (18), TNF-, and IL-1
(19) mRNA expression within the renal cortex has been
reported in MRL-lpr/lpr mice suffering from nephritis.
This line of evidence, along with the fact that G-CSF is already administered to lupus patients to alleviate neutropenia (20), prompted us to investigate whether the reported pro-Th2 and antiinflammatory properties of G-CSF might be of therapeutic benefit in the treatment of lupus disease. Therefore, we evaluated the effect of different dose regimens of G-CSF on the development of the lupus syndrome in MRL-lpr/lpr mice.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Six-week-old female MRL-lpr/lpr mice were purchased from Harlan Olac (Oxon, U.K.) and maintained in our animal facilities under specific pathogen-free conditions. Animals were randomly distributed into the different treatment groups (n = 12 per group).
Treatment of MRL-lpr/lpr/lpr mice with recombinant human G-CSF
Recombinant human G-CSF (Amgen, Thousands Oaks, CA) was injected s.c. every 6 wk for five consecutive days, starting at 9 wk of age. Mice at this age had no albuminuria. They had detectable anti-nucleosome but no anti-dsDNA Abs, typical of the onset of the autoantibody response in young lupus mice (21). Two doses of G-CSF were used: 10 µg/kg and 200 µg/kg, reported to protect C57BL/6 mice from GVHD (8) and elicit stem cell mobilization (22), respectively. The control group received the carrier solution (5% dextrose in sterile H20).
A second protocol consisted in a single late treatment with 200 µg/kg of G-CSF for five consecutive days at 13 wk of age, when mice presented high levels of circulating anti-nucleosome Abs with occasional low levels of anti-dsDNA Ab and emerging albuminuria.
Assessment of renal disease
Renal disease was evaluated by the development of albuminuria and histological modifications of the kidney. Albuminuria was measured colorimetrically using commercially available sticks (Albym-Test, Boehringer Mannheim, Mannheim, Germany).
Albumin concentrations over 1 mg/ml were considered positive. Kidney histology was performed on mice that were killed at 20 wk of age. The kidneys were removed, fixed in 3.6% paraformaldehyde in PBS, and included in paraffin. Sections of 5 µm were stained with hematoxylin-eosin and analyzed for glomerulonephritis. Evaluation of the Ig deposits in the glomeruli was performed on kidneys frozen in OCT (Tissue Tek-Miles, Elkart, IN). Fresh cryostat sections of 5 µm were fixed in acetone for 10 min, air dried, and saturated with 10% goat serum (Vector, Burlingame, CA) in PBS for 30 min at room temperature. After three washes, they were incubated for 30 min at room temperature with FITC-conjugated rat anti-mouse IgG3 (or anti-IgG2a) Ab (Southern Biotechnology Associates, Birmingham, AL), diluted 1/100 in PBS, supplemented with 1% goat serum. Stained sections were observed with a conventional fluorescence microscope.
Immunohistochemical detection of Fc receptors on kidney sections
Acetone-fixed cryostat sections (5 µm) were washed twice and
then incubated with 2 µg of 2.4G2 mAb (produced in our laboratory
from hybridoma) for 60 min. After three washes, biotinylated
anti-rat Ab (Zymed Laboratories, San Francisco, CA) was added for
15 min, sections were washed three times, and HRP-coupled streptavidin
(Zymed), with 3-amino-9 ethyl carbazole (Sigma, St. Louis, MO) as
substrate, was added to reveal FcRII (CD32) and FcRIII (CD16)
expression. Stained sections were observed under a light
microscope.
Detection of serum anti-nucleosome and anti-dsDNA autoantibodies
Serum autoantibodies were quantified by ELISA every 3 wk, as
previously described (21) with modifications as follows.
Briefly, microtiter plates were coated with nucleosomes (kindly donated
by Dr. Sophie Koutouzov, Institut National de la Santé et de la
Recherche Médicale Unité 25, Paris, France) at 5 µg/ml,
prepared as by Koutouzov et al. (23), or with
bacteriophage 
dsDNA (Boehringer, Mannheim, Germany) at 3.75 µg/ml
on poly-L-lysine-treated plates, by overnight incubation at
4°C. Sera were serially diluted, and isotypes of IgG specific for the
respective autoantigens were detected using specific anti-mouse
isotypes as secondary Abs including anti-IgG1, anti-IgG2a,
anti-IgG2b, and anti-IgG3 Abs coupled to HRP (Southern
Biotechnology Associates) at 1/1000 dilutions. Ab binding was measured
after the addition of 2,2'azino-di-3-ethyl benzothiazoline sulfonate
(Boehringer Mannheim) substrate at 0.3 mg/ml in citrate buffer, pH 4.0,
and H2O2 0.03%. The
reaction was stopped by the addition of sodium azide when intense color
developed. The results are expressed in absorbance units measured at
405 nm, with a reference filter at 550 nm. Values were extrapolated for
a nondiluted serum and adjusted for interplate and interassay
variations using a reference standard of pooled sera from 1-year-old
MRL-MP+/+ mice. Serum from adult C3H mice was
also included as a negative control. Titration curves using standard
IgG isotypes showed that an equal sensitivity was obtained across
isotypes, therefore allowing us to deduce total IgG production by
adding ODs obtained for individual isotypes.
Cytokine concentrations in serum
Amounts of total IL-12 (sera diluted at 1/8 and 1/16), IL-10,
TNF-, and soluble TNF receptors (TNF-sR55 and TNF-sR75) (sera
diluted 1/2) were measured with commercial ELISAs from Genzyme
(Cambridge, MA), according to the manufacturer’s instructions.
Cytokine production by splenocytes
At 20 wk of age, three mice per group were sacrificed and their
spleens, femurs, and tibias were removed. Spleen cells were enumerated
and CD4+ cells positively selected with
anti-CD4-labeled magnetic minibeads (Miltenyi Biotec,
Bergisch-Gladbach, Germany) using the magnetic cell separation system
technology. CD4+ cells (
95% pure, as assessed
by flow cytometry) were incubated at 2.105 cells
in a final volume of 100 µl/well in anti-CD3 (10 µg/ml, 50
µl)-coated 96-well culture plates in a humidified atmosphere at 5%
CO2. After 24 h, the supernatants were
removed and stored at -80°C until cytokine assays.
The following pairs of mAbs (prepared in our laboratory from hybridoma
or purchased from PharMingen (Becton Dickinson, Le Pont de Claix,
France)) were used for the ELISA measurements: AN 18 (2 µg/ml) and
R46A2 (0.5 µg/ml) for IFN-, 11B11 (2 µg/ml) and BVD6 (0.25
µg/ml) for IL-4, JES2.A5 (5 µg/ml) and JES5-16E3 (0.5 µg/ml) for
IL-10, for capture and detection, respectively. Bound secondary Abs
were detected with peroxidase-coupled streptavidin from Amdex
(Amersham, Buckingham, U.K.) for IL-4 and IL-10 and from Vector
Laboratories (Burlingame, CA) for IFN-, with
o-phenylene-diamine dichloride (Sigma) as substrate. ODs
were measured at 490 nm with a reference filter at 630 nm using a
Dynatech microplate reader (Dynex Technologies, Issy les Moulineaux,
France).
TNF- was measured with a commercial ELISA from Genzyme.
FACS analysis of spleen and bone marrow cells
The cell-surface phenotype of splenocytes and bone marrow cells
was analyzed by flow cytometry. All cells were incubated in PBS
supplemented with 2% FCS and 0.02% sodium azide. One million cells
per sample were incubated in 20 µl for 15 min at room temperature
under constant shaking. The following mAbs were used: anti-CD4
(clone GK1.5), anti-CD8 (clone 53-6.7), anti-ß TCR (clone
H57-597), anti-B220 (clone RA3-6B2), anti-Mac1 (clone M1/70),
and anti-GR-1 (clone RA3-8C5). They were produced in our laboratory
from hybridomas or purchased from PharMingen and used coupled to biotin
and revealed by streptavidin-tricolor (Caltag, South San Francisco,
CA), PE, or FITC.
For general phenotypic analysis, 10,000 events were acquired using the FACScan flow cytometer (Becton Dickinson, San Jose, CA). Analysis of the acquired data was performed using Lysis II software (Becton Dickinson).
In vitro colony-forming assay
Total CFU-C were quantified in "Complete Methylcellulose Medium with Recombinant Cytokines and Erythropoietin" (MethoCult GF M3434, Stem Cell Technologies, Vancouver, Canada). Cells were plated in a final volume of 1 ml at concentrations ranging from 5–50 x 104 cells per culture dish (Falcon 1008, Becton Dickinson, Lincoln Park, NJ). Colonies were scored on day 7–8.
Statistical analysis
Differences in serological parameters between the different
treatment groups were evaluated with Student’s t test. The
occurrence of mortality and albuminuria in the different treatment
groups were plotted using the Kaplan-Meier method, i.e., nonparametric
cumulated survival plots. The statistical comparison between the
different curves was performed using the Mantel-Cox log rank test,
which provided the corresponding 
2 values.
Values of p < 0.05 were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In control MRL-lpr/lpr mice, the onset of albuminuria
occurred at the age of 13 wk. Fifty percent of the animals were
positive at 19 wk and 100% at 25 wk (Fig. 1
A). The mortality rate
reached 50% in 23-wk-old mice (Fig. 1B).
|
In contrast, repeated administration of low-dose G-CSF at 10 µg/kg per injection markedly accelerated the onset of albuminuria, which was already detected at the age of 15 wk in one mouse of two, i.e., 1 mo earlier than in the excipient group. Mice died rapidly in this group (80–90% at 20 wk of age).
Renal histology and glomerular Ab deposits in high vs low-dose G-CSF-treated MRL-lpr/lpr mice
Kidneys from 20-wk-old mice representative for each treatment
group were collected and sectioned. For light microscopy, the
histologic slides were routinely processed and stained with
hematoxylin-eosin by standard procedures (Fig. 2, a–c). In the control group
(Fig. 2a), the lesions, essentially localized in the cortex,
revealed a moderate endoproliferative glomerulonephritis with initial
crescent formation and deposits along the capillary walls, "wire
loops." Almost intact glomeruli were observed in kidneys from mice
treated with 200 µg/kg of G-CSF (Fig. 2b), the group that
was significantly protected from disease. By contrast, mice injected
with G-CSF at 10 µg/kg (Fig. 2c) had a severe renal
disease, consisting of a diffuse proliferative and crescentic
glomerulonephritis with widespread "wire loops" and fibrinoid
necrosis, interstitial and perivascular inflammatory cells, and
moderate tubulo-interstitial abnormalities. Neither vasculitis nor
thrombotic microangiopathy were detected in any of the three
groups.
|
, d–f) was slightly weaker in
the kidney of mice chronically injected with 200 µg/kg of G-CSF (Fig. 2e) than in control mice (Fig. 2d). Conversely,
IgG2a deposits were present at similar levels in both treated and
control mice (not shown). In mice treated with 10 µg/kg of G-CSF,
IgG3 deposits (Fig. 2f) were much increased relative to the
control group, as were IgG2a (not shown). Inflammatory cytokine levels are reduced by treatment with high-dose G-CSF
Inflammation is a hallmark of disease progression in MRL-lpr/lpr mice. TNF soluble receptors (TNF-sR55 and TNF-sR75) represent stable and sensitive markers of an ongoing inflammatory reaction, and their levels are increased in the circulation of relapsing lupus patients (24, 25). In addition, IL-12 has been detected in the circulation of MRL-lpr/lpr mice but not in MRL+/+ mice, which develop only mild and late nephritis, or in nonautoimmune BALB/c mice (15).
As can be seen in Fig. 3A, the
two soluble TNF receptors were increased in the serum of control
MRL-lpr/lpr mice between 14 and 20 wk of age but did not
significantly change with age (particularly TNF-sR55) in mice subjected
to the high-dose G-CSF regimen. At 20 wk of age (Fig. 3B),
serum IL-12 was significantly decreased in MRL-lpr/lpr mice
that had been treated chronically with G-CSF at 200 µg/kg.
|
Serum levels of IL-10 (6.4 ± 4.3 pg/ml vs 9.3 ± 1.7 pg/ml
and 6.6 ± 2.8 pg/ml, for the excipient vs G-CSF 10 µg/kg and
200 µg/kg groups, respectively) at 20 wk of age, were not
significantly different between the three groups of mice. Any possible
increase in TNF- levels measured at the same age had no relationship
to disease outcome (10.45 ± 3.9 pg/ml vs 25.4 ± 13 pg/ml
and 27.2 ± 16 pg/ml, for the excipient vs G-CSF 10 µg/kg and
200 µg/kg groups, respectively) .
G-CSF-treatment modulates the glomerular expression of FcR in
MRL-lpr/lpr mice
Knowing that G-CSF modulates FcR expression on neutrophils and
macrophages (26) and that FcRIII (CD16) is expressed by
mesangial cells and mediates the inflammatory response to
im- mune complexes (27), we examined the glomerular
expression of CD16 in kidneys of mice from the different treatment
groups. Immunohistochemical staining of glomeruli (Fig. 2g)
from 20-wk-old control mice with the 2.4G2 Ab revealed Fc(RII+RIII)
receptors expression on infiltrating inflammatory cells as well as
mesangial cells. Interestingly, the well-preserved, noninflammed
glomeruli from mice treated with G-CSF at 200 µg/kg (Fig. 2h) were practically negative for 2.4G2 staining,
contrasting with the increased staining exhibited by mice treated with
low-dose G-CSF (Fig. 2i). This was confirmed using a
semiquantitative score to evaluate in a blinded fashion the intensity
of labeling of at least 30 glomeruli per kidney section for each of
three mice per group (Fig. 2j).
Effect of G-CSF on autoantibody production and isotype balance
The humoral autoimmune response in treated mice was evaluated both quantitatively, by measuring circulating autoantibody levels, and qualitatively, by determining their isotype profile.
As shown in Fig. 4, excepting a lower
mean level of circulating anti-nucleosome IgG2a isotype
(p < 0.05, relative to controls), no
significant changes in isotype ratio (calculated as the mean of
individual ratios) were detectable in the high-dose G-CSF group when
compared with excipient controls, whether the anti-nucleosome (Fig. 4A) or the anti-dsDNA (Fig. 4B) response was
considered. Total circulating anti-nucleosome IgG of all four
isotypes were reduced by 50% as compared with the control group,
whereas those directed against dsDNA did not significantly differ from
controls.
|
A). Concomitantly, a 10-fold increase in IgG1 levels
occurred relative to the control group, resulting in a dramatic
reduction of IgG2a/IgG1 and IgG3/IgG1 ratios. These changes were
detectable at 17 wk of age, i.e., 8 wk after the first treatment
period, and remained stable afterward (not shown). The sum of all four
isotypes of circulating anti-nucleosome IgG Abs was reduced by
30%. Interestingly, this marked switch in the isotype profile was
confined to the anti-nucleosome Ab response, as no such changes
could be detected in the anti-dsDNA response (Fig. 4B)
at 20 wk of age and/or later. However, total anti-dsDNA activity
comprising all four isotypes was 2-fold increased in mice treated with
10 µg/kg of G-CSF. None of the groups of mice exhibited significant differences in circulating levels of total IgG of each of the four isotypes (not shown).
Effect of G-CSF treatment on cellularity and on the cytokine profile in MRL-lpr/lpr mice
At 20 wk of age, i.e., 4 wk after the last series of G-CSF injections, when differences in the disease incidence were maximal between the three groups of mice, cellular analysis in spleen and bone marrow was performed at the same time as cytokine assays.
Changes in total spleen cellularity are depicted in Table I. Double negative (DN) T cells, typical of
the lpr phenotype, were reduced in both G-CSF treated
groups. Total spleen cell numbers were reduced (-45%) in mice treated
with low-dose G-CSF, but were unchanged in the group treated with
high-dose G-CSF, in which Mac-1+ cells had
considerably expanded (+70%). No significant changes were observed in
the granulocyte population recognized by the GR-1 Ab. In the bone
marrow, both cellularity and incidence of phenotypically defined cell
subsets were similar to those of excipient controls. The frequency of
spleen and bone marrow progenitors forming colonies in methylcellulose
remained also unchanged at this particular time point, 4 wk after
cessation of treatment (data not shown).
|
), no
modification of the cytokine profile in response to anti-CD3 was
seen when G-CSF was administered at 200 µg/kg per injection. However,
the low-dose group displayed a significant decrease in IFN-
production and a tendency, although not statistically significant, to
generate more IL-4. TNF- production was also reduced but IL-10
remained unaffected.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we demonstrate that chronic treatment of
lupus-prone MRL-lpr/lpr mice with G-CSF at 200 µg/kg
exerted a significant protection from nephritis. The TNF-/IL-10
balance in the circulation, which has been reported to be a determinant
for the disease progression in (NZB x
NZW)F1 mice (28), was not affected
by administration of G-CSF to MRL-lpr/lpr mice. Instead, the
reduced serum levels of IL-12 in 20-wk-old mice and the fact that
soluble TNF receptors did not increase with age, but remained at
predisease levels highly suggest that antiinflammatory effects of G-CSF
were responsible for the protection from nephritis observed in the
high-dose treatment. Moreover, the absence of changes in the Th1/Th2
balance in the autoreactive response of the mice injected with these
high doses of G-CSF indicates that the antiinflammatory effect was
exerted locally, at the glomerular level. At 20 wk of age, kidney
histology revealed almost intact glomeruli in treated mice, despite the
presence of immune complexes. This observation is reminiscent of the
uncoupling of immune complex deposits and kidney damage observed in
(NZB x NZW)F1 mice deficient for the
-chain of the Fc receptor (29). Interestingly, these
-/- animals, even with significant
proteinuria, did not progress to renal failure (29), a
feature shared with our group of high-dose G-CSF-treated mice (Fig. 1).
The binding of immune complexes to FcRIII (CD16) expressed by
mesangial cells triggers an inflammatory reaction (production of IL-6,
TNF-, and IL-12 in the renal cortex) (18, 27, 30) and
contributes to glomerular injury that leads to nephritis. The absence
of staining by 2.4G2 Ab (which recognizes FcRII and FcRIII) in
these kidneys suggests that the protective effect of G-CSF may involve
a decrease in FcRIII (CD16) expression on mesangial cells, similar
to the effect seen on neutrophils and macrophages (26),
where G-CSF induces the shedding of membrane CD16 in its soluble form
but has no effect on FcRII (CD32). Interestingly, soluble CD16 has
been shown to be beneficial in the treatment of murine lupus nephritis
(31). In addition, a reduced mesangial proliferation was
also observed, which may further contribute to the long-term reduction
in 2.4G2 expression induced by high-dose G-CSF treatment.
The paradoxical effect of the low-dose treatment with G-CSF represents
a particularly interesting aspect of our study. Indeed, treatment with
G-CSF at 10 µg/kg has been shown to protect from GVHD in C57BL/6 mice
by inducing a shift toward the Th2 phenotype (8), a model
from which, unfortunately, no information is available at high doses.
In keeping with this observation, we found that this low dose of growth
factor induced a decrease in IFN- production, a relative increase in
IL-4 secretion, and a corresponding shift in the IgG2a/IgG1 isotype
balance of the anti-nucleosome response in lupus-prone
MRL-lpr/lpr mice. Nevertheless, in Th2-oriented
MRL-lpr/lpr mice, the disease progress was markedly
accelerated. This result could be explained by the participation of
both Th1- and Th2-derived cytokines in the disease process, a notion
that is emerging from an increasing body of evidence obtained in murine
lupus models (9, 10, 11, 14, 17). The present results provide
further support to the above-cited evidence that a shift toward Th2 is
not necessarily protective in autoimmunity and suggest that
autoimmunity should not be reduced to a mere imbalance between Th1 and
Th2 cytokines. In particular, it should be taken into account that the
evolution of the renal disease is subject to a balance between
production and glomerular trapping and/or deposition of specific
isotypes of the autoantibodies. Indeed, immunofluorescence studies of
kidney sections demonstrated that the aggravation of lupus disease in
the low-dose group of G-CSF-treated mice corresponded to enhanced Ig
deposits, particularly of the nephritogenic IgG3 and IgG2a isotypes,
that might be explained by an increased glomerular expression of
FcRI induced by G-CSF (26).
Interestingly, IgG isotype changes concerned only the autoimmune response and specifically the anti-nucleosome activity, whereas the anti-dsDNA activity remained unchanged. The production of anti-nucleosome Abs that recognize quaternary epitopes of the Ag (21, 32) precedes that of anti-dsDNA in MRL-lpr/lpr mice. Nucleosome-specific T cells are able to accelerate disease progression in (NZB x SWR)F1 mice (33), suggesting that the nucleosome particle, but not its individual components, is the primary T cell Ag in lupus. Therefore, the Th1/Th2 balance of T cell-derived cytokines may preferentially influence the isotype profile of anti-nucleosome Ab production. In addition, studies in lupus patients have recently demonstrated that increased Ab levels of specific isotype during disease relapses were observed only among anti-nucleosome, but not among anti-ssDNA or anti-dsDNA, Abs (Z. Amoura, unpublished observations).
The effect of G-CSF administration on spleen and bone marrow cellularity and phenotype was analyzed in 20-wk-old mice. This time point was chosen because the differences in disease progression between control and treated mice were the most striking. The treatment with G-CSF had been interrupted 4 wk before, which might explain why the numbers of myeloid progenitors and GR1+ granulocytes was not modified. This observation is in agreement with the previously reported transient effect of G-CSF on neutrophil and progenitor cell mobilization (22, 26, 34, 35). The increase in the Mac-1+ population, which is known to be already expanded in MRL-lpr/lpr mice (36), indicates that G-CSF has nevertheless exerted some hemopoietic effect. Interestingly, as previously noted for pluripotent stem cells in other autoimmune strains (37), the number of progenitors forming colonies in methylcellulose was remarkably high in spleens of MRL-lpr/lpr mice (97,123.8 ± 2,131.0; mean ± SEM from six mice), as compared with age-matched MRL-MP+/+ controls (26,074.3 ± 7,177.6; mean ± SEM from six mice; p < 0.05), possibly hampering their further expansion in response to G-CSF.
Both treatment regimens significantly reduced
TCRß+
CD4-CD8- DN cell numbers.
It has been documented that the accumulation of DN T cells in spleen
and lymph nodes is clearly dissociated from the autoimmune renal
disease (38, 39, 40). The opposite disease outcome promoted by
the high- and low-dose treatment with G-CSF, in spite of a similar
reduction in this DN population, confirms that DN cells play no
pathogenic role in lupus-prone MRL-lpr/lpr mice. However, it
remains to be established whether this apparent decrease of DN cells is
effectively due to a reduction or a redistribution to other sites such
as liver or peritoneal cavity.
Our data provide the first evidence for a therapeutic impact of hemopoietic growth factors on autoimmune diseases. The protection against lupus nephritis by antiinflammatory doses of G-CSF may hold promise for a clinical evaluation of this factor in lupus patients. Our finding that a 5-day treatment with G-CSF at 200 µg/kg, initiated in 13-wk-old mice suffering already from a beginning proteinuria, still delayed the disease onset, suggests that chronic administration of G-CSF might be beneficial for lupus patients suffering from nephritis, i.e., even at a relatively advanced stage of the disease.
In addition to their fundamental interest, our results are of clinical relevance because G-CSF is already administered to lupus patients to relieve neutropenia, which is a common complication of the lupus syndrome. This is well illustrated by the reports showing that G-CSF efficiently restores normal neutrophil counts in lupus patients. Yet, in the light of our data, the dose of G-CSF injected has to be chosen with caution. Indeed, vasculitis and aggravation of neurolupus were observed in one and two of nine patients treated with G-CSF, respectively (20). Finally, recent clinical trials using G-CSF-mobilized autologous stem cell transplantation in patients with autoimmune diseases, including lupus patients (41), have provided interesting results. Therefore, it is even more important to elucidate the dose-dependent effect of G-CSF on the progression of lupus disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: GVHD, graft-versus-host disease; DN, double negative.
Received for publication May 7, 1999. Accepted for publication August 23, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Immunol. 149:918.[Abstract]
is essential for the development of autoimmune glomerulonephritis in MRL/lpr mice. J. Immunol. 158:5484.[Abstract]
is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice. J. Clin. Invest. 101:364.[Medline]
and interleukin-4 in murine lupus. J. Clin. Invest. 99:1936.[Medline]
interferon. J. Exp. Med. 166:798. receptor inhibits the onset of glomerulonephritis. Eur. J. Immunol. 25:6.[Medline]
RIII-a) and the associated Fc
RI -chain. J. Immunol. 153:1281.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  A.-M. Fairhurst, C. Xie, Y. Fu, A. Wang, C. Boudreaux, X. J. Zhou, R. Cibotti, A. Coyle, J. E. Connolly, E. K. Wakeland, et al. Type I Interferons Produced by Resident Renal Cells May Promote End-Organ Disease in Autoantibody-Mediated Glomerulonephritis J. Immunol., November 15, 2009; 183(10): 6831 - 6838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Gaudreau, C. Guindi, M. Menard, G. Besin, G. Dupuis, and A. Amrani Granulocyte-Macrophage Colony-Stimulating Factor Prevents Diabetes Development in NOD Mice by Inducing Tolerogenic Dendritic Cells that Sustain the Suppressive Function of CD4+CD25+ Regulatory T Cells J. Immunol., September 15, 2007; 179(6): 3638 - 3647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Burt, A. Traynor, L. Statkute, W. G. Barr, R. Rosa, J. Schroeder, L. Verda, N. Krosnjar, K. Quigley, K. Yaung, et al. Nonmyeloablative Hematopoietic Stem Cell Transplantation for Systemic Lupus Erythematosus JAMA, February 1, 2006; 295(5): 527 - 535. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Teuscher, R. W. Doerge, P. D. Fillmore, and E. P. Blankenhorn eae36, a Locus on Mouse Chromosome 4, Controls Susceptibility to Experimental Allergic Encephalomyelitis in Older Mice and Mice Immunized in the Winter Genetics, February 1, 2006; 172(2): 1147 - 1153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rutella, F. Zavala, S. Danese, H. Kared, and G. Leone Granulocyte Colony-Stimulating Factor: A Novel Mediator of T Cell Tolerance J. Immunol., December 1, 2005; 175(11): 7085 - 7091. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kared, A. Masson, H. Adle-Biassette, J.-F. Bach, L. Chatenoud, and F. Zavala Treatment With Granulocyte Colony-Stimulating Factor Prevents Diabetes in NOD Mice by Recruiting Plasmacytoid Dendritic Cells and Functional CD4+CD25+ Regulatory T-Cells Diabetes, January 1, 2005; 54(1): 78 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zou, B. Barnett, H. Safah, V. F. LaRussa, M. Evdemon-Hogan, P. Mottram, S. Wei, O. David, T. J. Curiel, and W. Zou Bone Marrow Is a Reservoir for CD4+CD25+ Regulatory T Cells that Traffic through CXCL12/CXCR4 Signals Cancer Res., November 15, 2004; 64(22): 8451 - 8455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhang, V. K. Woodward, J. M. Shelton, J. A. Richardson, X. J. Zhou, D. Link, M. L. Kielar, D. R. Jeyarajah, and C. Y. Lu Ischemia-reperfusion induces G-CSF gene expression by renal medullary thick ascending limb cells in vivo and in vitro Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1193 - F1201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Zavala, S. Abad, S. Ezine, V. Taupin, A. Masson, and J.-F. Bach G-CSF Therapy of Ongoing Experimental Allergic Encephalomyelitis Via Chemokine- and Cytokine-Based Immune Deviation J. Immunol., February 15, 2002; 168(4): 2011 - 2019. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Tschetter, E. Mozes, and G. M. Shearer Progression from Acute to Chronic Disease in a Murine Parent-into-F1 Model of Graft-Versus-Host Disease J. Immunol., November 15, 2000; 165(10): 5987 - 5994. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 10 µg/kg (
), or excipient (
). Albuminuria was examined
weekly using dip sticks (Fig. 1
