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by Macrophages Controls Their Recruitment to Kidney and the Development of Glomerulonephritis in MRL/lpr Mice1
* Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientificas, Cantoblanco, Madrid, Spain; and
Departamento de Patología Animal II, Facultad de Veterinaria, Universidad de Complutense, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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+/- MRL/lpr mice
which produce high anti-DNA Ab levels but are protected from kidney
disease. The results showed that defective macrophage recruitment to
IFN-+/- mouse kidney was not caused by decreased levels
of monocyte chemoattractant protein-1, a chemokine that controls
macrophage migration to MRL/lpr mouse kidney. To
determine which IFN--producing cell type orchestrates the
inflammation pathway in kidney, we transferred IFN-+/+
monocyte/macrophages or T cells to IFN--/- mice, which
do not develop anti-DNA autoantibodies. The data demonstrate that
IFN- production by infiltrating macrophages, and not by T cells, is
responsible for adhesion molecule up-regulation, macrophage
accumulation, and inflammation in kidney, even in the absence of
autoantibody deposits. Therefore, in addition to monocyte
chemoattractant protein-1, macrophage-produced IFN- controls
macrophage migration to kidney; the degree of IFN- production by
macrophages also regulates glomerulonephritis development. Our findings
establish the level of IFN- secretion by macrophages as a link
between anti-DNA autoantibody deposition and glomerulonephritis
development, outline the pathway of the inflammatory process, and
suggest potential treatment for disease even after autoantibody
development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain thus
exhibit glomerular immune complexes (IC), but not glomerulonephritis,
presumably by disruption of the FcR-mediated inflammatory cascade
(1). We also observed protection from kidney disease in
IFN-+/- MRL/lpr mice, despite
intact autoantibody production and IC deposition (2). A
similar model of delayed disease has been described for monocyte
chemoattractant protein (MCP)-1-deficient MRL/lpr
mice (3).
IFN- is a key cytokine that controls Th1-dependent Ab production and
is indispensable for functional immune responses (4, 5).
The elevated IFN- expression in MRL/lpr mice (6, 7), the exacerbating effect on disease onset in NZB x NZW mice
following treatment with this cytokine (8), and the
amelioration of disease manifestations following inactivation of
IFN- (8, 9) suggest an important role for this cytokine
in lupus development. Nevertheless, the mechanism by which IFN-
contributes to this autoimmune disease remains unclear. Deletion of
IFN- or its receptor in MRL/lpr mice demonstrated that
this cytokine is essential for autoantibody production (2, 10, 11, 12, 13), a result further confirmed in a myasthenia gravis model
(14). Complete absence of IFN- is not required to
ameliorate disease; severe reduction of IFN-, mediated by gene
therapy, affects both autoantibody production and glomerulonephritis
development (15). A presumably less efficient IFN-
decrease, detected in a long-lived MRL/lpr mouse mutant,
results in change in autoantibody isotype and enhances survival
(16). An 
50% decrease in IFN- levels in
IFN-+/- MRL/lpr mice results in
greatly delayed disease onset (2). The elevated
autoantibody and IC deposition levels in
IFN-+/- mice, similar to those of
wild-type (wt) MRL/lpr mice, suggest that IFN-
levels play a key role in the local inflammatory process.
Initiation of the kidney inflammatory process is thought to depend on
autoantibody deposition, which ultimately leads to tissue destruction,
but the underlying mechanism is not known. Analysis of MCP-1-deficient
MRL/lpr mice showed that MCP-1 controls the inflammatory
process in kidneys of lupus-prone mice by inhibiting macrophage
migration and delaying disease onset, despite elevated IC deposits in
the kidneys of these animals (3). Therefore, MCP-1 is
required for local kidney inflammation, although IFN- may also exert
an effect on this process.
To assess the role of IFN- in kidney inflammation, we studied
factors that may influence this process in
IFN-+/- and in
IFN--/- MRL/lpr mice following
transfer of IFN--producing monocyte/macrophages or T cells. The data
showed that IFN- secretion by macrophages controls their recruitment
to kidney interstitium, up-regulation of the adhesion molecules ICAM-1
and VCAM-1, as well as the overall inflammatory process. Furthermore,
efficient IFN- production by macrophages is indispensable for
disease progression in the presence of IC glomerular deposits.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously described the generation of
IFN--/- MRL/lpr mice
(2). In this study, we used
IFN--/-, IFN-+/-,
and IFN-+/+ mice. Mice were typed by PCR using
appropriate oligonucleotides (2).
Immunostaining
To detect IC deposits, cryostat kidney sections (6 µm) were fixed in chilled acetone and stained with a FITC-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL). Immunostaining was also performed with biotin-conjugated Abs to CD4, CD8 (Caltag Laboratories, Burlingame, CA), VCAM-1 (BD PharMingen, San Diego, CA), and F4/80 (Serotec, Oxford, U.K.). MCP-1 was detected using a goat anti-mouse MCP-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA), followed by peroxidase (PO)-conjugated donkey anti-sheep/goat secondary Ab (The Binding Site, Birmingham, U.K.). Staining was performed as described (2). Endogenous peroxidase activity was blocked by incubation with PBS containing 0.03% H2O2. For staining with biotin-conjugated Abs, sections were incubated with a biotin blocking kit (DAKO, Glostrup, Denmark) and 20% normal mouse serum. Biotinylated Abs were developed with the StrepABComplex/HRP kit (DAKO). When anti-MCP-1 Ab was used, sections were blocked with 20% normal horse serum. Peroxidase activity was detected using FAST DAB (Sigma-Aldrich, St. Louis, MO).
For paraffin sections, kidneys were fixed in 5% formalin, 4-µm sections were deparaffinized and treated with 3% H2O2 in 70% methanol, followed by microwave treatment in 10 mM sodium citrate buffer. Biotin block and serum treatment was performed as above. ICAM-1 expression was detected using a biotin-conjugated Ab (BD PharMingen); MCP-1 was detected using goat anti-mouse MCP-1 Ab. CD3 was identified by a rabbit anti-human CD3 Ab, followed by PO-labeled goat anti-rabbit Ab (both from DAKO). PO-conjugated anti-proliferating nuclear Ag (PCNA; Santa Cruz Biotechnology) was used to stain paraffin sections according to manufacturer’s instructions, including blocking steps with H2O2 and 20% normal serum.
To evaluate the degree of F4/80, ICAM-1, and VCAM-1 expression, 10 random glomeruli and 10 tubular zones were evaluated from each section. Sections were scored on a scale of 1–4+; a score of 4+ was assigned to stained sections of a 4-mo-old MRL/lpr mouse with full-blown glomerulonephritis and maximal staining for F4/80, ICAM-1, and VCAM-1. Evaluation of degree of staining in sections from other mice were based on this mouse.
RNA protection assay
Kidney chemokines were detected by multiprobe RNase protection assay according to manufacturer’s instructions (BD PharMingen). Expression levels were quantitated as a function of L32 and GADPH housekeeping gene levels in a Storm 860 PhosphorImager and analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
In vivo 5-bromo-2'-deoxyuridine (BrdU) administration
Mice were given BrdU (0.8 mg/ml; Sigma-Aldrich) in drinking water, prepared freshly every 2 days, for a 9-day period.
Flow cytometric analysis and sorting
BrdU expression in macrophages was determined by triple staining
with FITC-conjugated anti-BrdU, PE-anti-Mac-1 (both from
BD PharMingen) and Cy5-anti-F4/80 (Serotec) Abs, as described
(2). To detect macrophage CCR2 expression, we used a
biotin-conjugated mouse anti-human CCR2 (17) that
cross-reacts with murine CCR2, followed by FITC-conjugated streptavidin
(Caltag Laboratories) in combination with PE-anti-Mac-1 and
Cy5-F4/80. For detection of macrophage IFN-R expression, a
biotinylated anti-IFN-R 
-chain Ab (BD PharMingen) was used in
combination with PE-anti-MAC-1 and Cy5-anti-F4/80 Abs. We used
these last two Abs and FITC-conjugated
anti-I-Ak (BD PharMingen) to determine MHC
class II expression levels. Stained cells were analyzed on a Coulter
EPICS XL flow cytometer (Hialeah, FL).
To purify monocytes/macrophages, splenocytes were stained with FITC-labeled Abs to CD4, CD8, and B220 (BD PharMingen) and passed through a MACS separation column (Miltenyi Biotec, Bergisch Gladbach, Germany) designed to retain FITC-labeled cells. Recovered cells were further stained with a PE-anti-Mac-1 Ab and sorted using a Coulter EPICS sorter; a yield of 99% pure Mac-1+ cells was obtained. CD4+ and CD8+ lymphocytes were also sorted following double-labeling with FITC- and PE-conjugated anti-CD4 and anti-CD8 Abs, respectively. To sort CD4+ and CD8+ T cells as well as macrophages from adoptively transferred hosts, splenocytes were stained with a combination of FITC-labeled Abs to CD4 and CD8, and with PE-anti-Mac-1 and Cy5-anti-F4/80 Abs.
RT-PCR
RNA was extracted from sorted CD4+ and CD8+ T cells (combined), macrophages, as well as from whole kidney. RT-PCR was performed using a OneStep RT-PCR kit (Qiagen, Hilden, Germany) using primers and PCR conditions previously described (2).
Serological and histological analyses
Isotype-specific anti-DNA Abs were determined by ELISA as
described (2). The reactivity of PO-conjugated Abs against
IgG1, IgG2, IgG2b, and IgG3 (Caltag Laboratories) was normalized for
equivalent OD against corresponding myeloma isotype controls (Caltag
Laboratories). Serum IFN- levels were quantified with a mouse
IFN- ELISA kit (Endogen, Woburn, MA) following manufacturer’s
instructions. Histological examination of lungs or kidneys was
performed in a blind manner; glomerulonephritis was scored on a scale
of 0–4+ and pyogranulomatous nephritis was evaluated as light,
moderate, or severe. Blood urea nitrogen (BUN) levels were determined
at autopsy using Azostix strips (Bayer, Elkhart, IN).
Statistics
The Mann-Whitney U test was used to evaluate statistical significance. Two-tailed p values of <0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+/- MRL/lpr mice
Using IFN--/- and
IFN-+/- MRL/lpr mice as models, we
attempted to define the factors that control kidney inflammation in
lupus nephritis. IFN- deficiency prevents anti-DNA Ab production
and disease onset (2). In contrast,
IFN-+/- MRL/lpr mice,
characterized by an 50% reduction in IFN- production, develop
elevated autoantibody levels, but are protected from glomerulonephritis
development (2). IFN-+/- mice
thus constitute a useful model for the study of the pathogenic
mechanism of kidney inflammation in SLE.
One characteristic of kidney disease in lupus-prone mice is
extensive inflammatory cell infiltration. We observed a
perivascular infiltrate, composed mainly of
CD4+ cells, in the kidneys of
IFN--/-, IFN-+/-,
and wt MRL/lpr mice (Fig. 1
A).
CD8+ cells represented a smaller proportion of
the infiltrating cells, with no apparent differences among the three
types of mice examined (not shown).
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, p < 0.002;
Fig. 1
B and not shown). Compared with wt MRL/lpr
mice, 4-mo-old IFN--/- mice lacked
kidney-infiltrating macrophages (Fig. 1B; Table I), a
finding compatible with the absence of IC deposits in the knock-out
mice and in agreement with similar findings in IFN-R-deficient mice
(13). Macrophage accumulation was also dramatically
reduced in IFN-+/- MRL/lpr mice
(Table I, IFN-+/- vs
IFN-+/+, p < 0.002; Fig. 1B). The absence of disease in these animals could thus be
associated with reduced macrophage presence in kidney.
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heterozygosity does not affect MCP-1 up-regulation in
MRL/lpr mouse kidney
Because MCP-1 is a critical molecule in leukocyte migration
to inflammation sites and lupus glomerulonephritis, we considered that
reduction in IFN- levels may influence macrophage migration by
affecting MCP-1 expression in kidney. We compared MCP-1, RANTES,
and macrophage-inflammatory protein (MIP)-1
expression
in kidneys of 4-mo-old IFN-+/+,
IFN--/- and
IFN-+/- MRL/lpr mice by
immunohistochemistry in paraffin sections. RANTES was uniformly
expressed by tubular cells, whereas MIP-1 was expressed solely by
distal tubules (not shown). MCP-1, which is also present in tubules,
shows a specific expression profile, with some tubular cells exhibiting
intense MCP-1 production oriented toward the luminal brush border (Fig. 1C). This expression pattern was similar in wt,
IFN-+/- and
IFN--/- MRL/lpr mice. Although
up-regulation of MCP-1 expression has also been detected in lupus-prone
mouse glomeruli by in situ hybridization (18),
glomerular MCP-1 expression was low in paraffin sections (Fig. 1C). Staining of kidney cryosections showed pronounced
glomerular MCP-1 expression in all three mouse types (Fig. 1D). The variation in staining between cryo- and paraffin
sections may depend on differences in accessibility of distinct kidney
structures, depending on the technique used.
Multiprobe RNase protection assay was used to quantitate the expression
of several chemokines. Kidney expression of RANTES, eotaxin, MIP-1,
and MCP-1 was similar in 3- and 4-mo-old MRL/lpr mice (Fig. 2A). This concurs with
immunohistochemical analysis, in which MCP-1 expression was similar in
3- and 4-mo-old mouse kidney (not shown) and with a previous report
showing equivalent MCP-1 expression in 2- and 4-mo-old mice
(3). One-month-old animals showed lower kidney MCP-1
levels compared with 3- and 4-mo-old animals. There was no difference
in chemokine levels between IFN-+/+ and
IFN-+/- animals (Fig. 2A); kidney
MCP-1 expression was lower in IFN--/- mice
(by 33%) compared with IFN-+/+ controls at 4
mo of age (p < 0.006, n = 10),
but clearly elevated compared with 1-mo-old mice
(p < 0.0001). This concurs with a previous
report in which reduced MCP-1 production was detected in
MRL/lpr mice subjected to gene therapy with cDNA encoding
IFN-R/Fc (15). IFN--inducible protein-10
levels were determined using a different probe set (not shown); this
chemokine was not up-regulated in MRL/lpr mouse kidney.
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+/-
MRL/lpr mice was not sufficient for macrophage attraction.
To exclude the possibility that this was due to an intrinsic macrophage
defect provoked by reduced IFN- production, we examined various
functional aspects of these cells. Macrophages (Mac-1+, F4/80+) were
represented in similar proportions in spleens of
IFN-+/+ and IFN-+/-
mice (12 ± 2 and 10 ± 1.2%, n = 6); there
were no differences in size and cellularity between the spleens of the
two mouse types. Macrophages from IFN-+/+ and
IFN-+/- animals proliferated similarly, as
assayed by BrdU incorporation (Fig. 2B). Because CCR2 is the
major MCP-1 chemokine receptor (21, 22, 23), we examined
whether expression of this receptor was IFN--dependent; there was no
difference in CCR2 expression in spleen macrophages from
IFN-+/+ and IFN-+/-
mice (Fig. 2C). Another indicator of macrophage activation
is the MHC class II expression level, which is up-regulated in
activated macrophages (5). We previously showed that
macrophage I-Ak expression is reduced in
MRL/lpr IFN--/- mice
(2). In contrast, in this study, we find similar
I-Ak expression in
IFN-+/+ and IFN-+/-
mouse macrophages (Fig. 2D). We thus show that decreased
IFN- production does not affect critical functional characteristics
of macrophages that could interfere with their migration to
inflammation sites.
Adhesion molecule up-regulation depends on the degree of
IFN- expression in MRL/lpr mice
ICAM-1 is an adhesion molecule involved in leukocyte attraction to
inflammation sites; it is also implicated in the progression of
lupus-associated glomerulonephritis (24). To assess the
possible implication of this molecule in the delayed disease in
IFN-+/- and
IFN--/- MRL/lpr mouse kidney, we
examined ICAM-1 expression by immunohistochemistry in 4-mo-old mice.
The data show elevated ICAM-1 expression in kidneys of 4-mo-old
MRL/lpr mice kidneys, but not in those of 4- or 6-mo-old
IFN--/- or IFN-+/-
animals (Fig. 3A; Table I,
IFN-+/+ vs IFN-+/-,
p < 0.002), indicating that ICAM-1 expression depends
on the degree of IFN- expression. In addition, the VCAM-1 adhesion
molecule, which is up-regulated in SLE glomerulonephritis
(24), appeared highly dependent on IFN- levels (Fig. 3B; Table I, IFN-+/- vs
IFN-+/+, p < 0.002). Finally,
kidney ICAM-1 and VCAM-1 expression was very low in 3-mo-old
MRL/lpr mice, whereas elevated expression was detected at 4
mo of age (Table I, p < 0.002 and p <
0.002, respectively). Because F4/80 cell migration takes place
in this interval, we conclude that adhesion molecule up-regulation
coincides with the appearance of F4/80+ cells in
kidney.
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regulates the kidney inflammatory
process
F4/80+ macrophages have an important role in
the progression of inflammation and lupus glomerulonephritis
development. Macrophage recruitment to the kidney is blocked, even in
the presence of heavy glomerular immune deposits in
IFN-+/- mice. Although MCP-1 is produced by
tubular cells and glomeruli, the site of IFN- production during
kidney inflammation is not clear. IFN- production by infiltrating
cells has been reported under conditions of increased IL-12 expression
(25). To identify the source of IFN--producing cells
responsible for macrophage attraction, we adoptively transferred
purified monocyte/macrophages from IFN-+/+ to
IFN--/- animals.
Two-month-old IFN--/- mice each received an
i.v. injection of 106 purified spleen
monocytes/macrophages from IFN-+/+ mice;
recipient animals (n = 9) and controls
(n = 7) were studied for 4 mo. Adoptively transferred
macrophages produced IFN- in IFN--/-
hosts, because at 4 mo posttransfer, IFN- mRNA was detected in
macrophages, but not in T cells, of recipient animals (Fig. 4A, left panel).
IFN- production was further confirmed by the ELISA detection of this
cytokine in the serum of transferred animals, at levels similar to
those of BALB/c mice but much lower than of MRL/lpr mice
(Fig. 4A, right panel). IFN-R expression by
macrophages confirmed their potential responsiveness to IFN- (Fig. 4B). Although some anti-DNA IgG1 production was detected
in recipient mice compared with age-matched
IFN--/- controls (13 ± 3.0 vs 5
± 0.7 OD U), anti-DNA Ab generation was minimal compared with that
of IFN-+/+ mice (110 ± 33,
n = 6). The autoantibody increase was detected
only for the IgG1 isotype (considered nonpathogenic; Ref.
16); IgG2a, IgG2b, and IgG3 levels were undetectable in
both adoptively transferred and control
IFN--/- mice. The amount of IFN- produced
was thus insufficient to provoke significant anti-DNA Ab production
or a switch to Th1-dependent isotypes.
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+/+ animals and
similar to IFN--/- controls (Fig. 4C). MCP-1 expression quantified by RNase protection assay
was similar in macrophage-transferred and
IFN--/- control animals (40 ± 14,
n = 8 vs 42 ± 17, n = 10 (Fig. 2A)). Concurring with these results, immunohistochemistry
experiments showed similar glomerular MCP-1 expression in recipient and
control mice (Fig. 4D). In overall agreement with these
results, we observed reduced glomerular proliferation in kidneys of
recipient mice compared with those of wt MRL/lpr mice, using
an Ab to PCNA (Fig. 5A);
disease-protected IFN--/- controls and
IFN-+/- mice also showed minimal glomerular
proliferation (Fig. 5A).
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+/+ monocyte/macrophages to
IFN--/- mice was sufficient for migration of
F4/80+ macrophages to the periglomerular and
peritubular areas of the kidney, even in the absence of high
autoantibody titers (Fig. 5B) and despite the 33% reduction
in kidney MCP-1 production (Fig. 2A) in the host mice.
Although IFN--deficient macrophages may also have migrated to the
kidney, IFN-+/+ macrophages accumulate in this
organ, because IFN- was expressed in host
IFN--/- animal kidneys (Fig. 4A,
left panel). CD4+ cells were detected
in the interstitial areas of kidneys in transferred mice, whereas
CD8+ and B cells were rarely identified
(not shown). In spleen, macrophages were found in similar proportions
in adoptively transferred animals and
IFN--/- controls (not shown).
Immunohistochemistry experiments showed increased ICAM-1 and VCAM-1
production in IFN--/- recipient mouse kidney
(Fig. 5B; Table I), emphasizing the contribution of
IFN--producing macrophages in adhesion molecule up-regulation. In
PCNA staining experiments, we detected no appreciable proliferation of
the interstitial infiltrate in wt MRL/lpr mice or adoptively
transferred IFN--/- animals (not shown).
This suggests that the interstitial macrophage accumulation is due to
migration and not to local proliferation. Finally, as an additional
control, IFN--/- mice were adoptively
transferred with IFN--/- macrophages
(n = 5) and their kidneys were compared with those of
control IFN--/- mice; there was no
macrophage accumulation in the kidneys of mice adoptively transferred
with IFN--/- macrophages (not shown). This
confirms that the IFN--producing capacity of macrophages is
responsible for their accumulation in the kidney.
Histological analysis of kidneys showed absence of glomerulonephritis
in IFN-+/+ macrophage-recipient mice, as is
the case for IFN--/- control mice. In the
adoptively transferred animals, however, kidneys showed severe
multifocal pyogranulomatous nephritis, with extensive perivascular and
periglomerular accumulation of polymorphonuclear leukocytes and
mononuclear cells. Degenerative glomerular lesions were observed, with
atrophy and some completely fibrotic glomeruli (Fig. 6A). Pyogranulomatous
nephritis was severe in all transferred animals (n = 9,
severity was classified on a scale of light, moderate, or severe
disease), while nephritis was absent in all control animals
(n = 7; Fig. 6A).
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does not affect macrophage-associated
kidney inflammation, but contributes to the inflammatory process in the
lung
To determine whether intact IFN- production by T cells could
initiate the inflammatory process in the kidney and recruit
F4/80+ cells, we adoptively transferred purified
IFN-+/+ T cells to
IFN--/- mice. Recipient mice were
2-mo old at the time of transfer of 106
CD4+ and 106
CD8+ T cells. Transferred and control animals
were followed for 4 mo after T cell transfer. As detected by RT-PCR of
purified CD4+ and CD8+
splenocytes at 4 mo posttransfer, adoptively transferred T cells
expressed IFN- (not shown). The results of
IFN-+/+ T cell transfer to
IFN--/- mice showed that the kidney
interstitium of these mice remained free of
F4/80+ cells, T cells, or other types of
lymphocytes, and showed low ICAM-1 and VCAM-1 levels (Fig. 5C; Table I). This indicates that
IFN-+/+ T cells, different from
IFN-+/+ macrophages, are not sufficient
to initiate local interstitial kidney inflammation (Fig. 5, B and C; Table I). Host mice developed heavy
perivascular infiltrates composed of CD4+ T cells
(not shown), as was the case for IFN--/-
mice (Fig. 1A). T cell-transferred mice showed no signs of
glomerulonephritis (not shown) and no glomerular proliferation (Fig. 5A), as was also the case for nontransferred controls. In
contrast, transferred mice developed diffuse severe acidophilic
pneumonia, characterized by intraalveolar accumulation of strongly
eosinophilic macrophages with heavy CD3+ cell
infiltration compared with IFN--/- controls
(Fig. 7);
IFN--/- animals reconstituted with
IFN-+/+ macrophages presented occasional
peribronchial T cell infiltration (Fig. 7). We also examined the
serological effects of IFN-+/+ T cell transfer
in IFN--/--recipient mice, and found that
transfer alone did not reconstitute anti-DNA autoantibody
production (12 ± 0.5 OD U in IFN--/-
transferred vs 7 ± 0.5 OD U in IFN--/-
controls). As lymph node and spleen hypercellularity and
hyperproliferation persist in IFN--/- mice
(2), the number of transferred
IFN-+/+ T cells may be insufficient to give
rise to anti-DNA autoantibody production. Alternatively, break of
tolerance to DNA may require intact IFN- production by both T cells
and macrophages.
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production by macrophages is sufficient for
glomerulonephritis development in the presence of glomerular
autoantibody deposits
Adoptive transfer of IFN-+/+
macrophages or T cells into
IFN--/--recipient mice shows that
intact IFN- production by macrophages is responsible for their
attraction to the kidney interstitium and establishment of the
inflammatory process. Nonetheless, glomerulonephritis did not develop
in the absence of glomerular deposits in
IFN--/- mice. To determine the role of
macrophage-produced IFN- in glomerulonephritis development, we
transferred IFN-+/+ macrophages
(106 cells/mouse) into 2-mo-old
IFN-+/- mice, which develop glomerular
autoantibody deposits, but not glomerulonephritis. We followed these
mice (IFN-+/- (M)) for 4 mo posttransfer and
observed that all transferred mice developed severe diffuse
proliferative glomerulonephritis with hypercellular glomerulus,
increased mesangial cells, and thickening of capillary walls (Fig. 6B). Transferred mice were sacrificed when they became
moribund (n = 4) or at 4 mo posttransfer
(n = 2), while all controls remained alive at this
time. Glomerular damage was significantly more severe in transferred
mice than in IFN-+/- controls (Table II); concurring with the
glomerulonephritis data, transferred animals showed elevated BUN levels
at death. The degree of glomerular damage and BUN levels in
IFN-+/- (M) mice was similar to 6-mo-old
IFN-+/+ controls. F4/80+
cell migration was restored, as was adhesion molecule up-regulation in
mice reconstituted with IFN-+/+ macrophages
(not shown). The data indicate that glomerulonephritis development in
IFN-+/- mice is dependent on the transfer of
IFN-+/+ macrophages, which potentiated the
inflammatory pathway and resulted in glomerular damage. Glomerular
autoantibody deposition and intact IFN- production by macrophages
are thus two events sufficient for glomerulonephritis development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+/-
MRL/lpr mice, which are protected from glomerulonephritis,
and in Ab-free IFN--/- mice
adoptively transferred with IFN-+/+
monocyte/macrophages or T cells. The following conclusions are drawn as
a result of this study: 1) impaired IFN- production in
IFN-+/- MRL/lpr mice results in a
remarkable decrease in interstitial F4/80+
macrophages, but does not affect the elevated MCP-1 expression in the
MRL/lpr mouse kidney, 2) adhesion molecules may be essential
for macrophage accumulation, because ICAM-1 and VCAM-1 expression is
substantially reduced in IFN-+/-
MRL/lpr mouse kidneys, 3) IFN- production by macrophages,
but not by T cells, is sufficient to initiate adhesion molecule
up-regulation in the kidney and further macrophage recruitment, even in
the absence of glomerular autoantibody deposits, and 4) in addition to
anti-DNA autoantibody deposition, intact IFN- production by
macrophages is necessary for glomerular destruction.
Several studies implicate MCP-1 in the attraction of inflammatory cells
to sites of tissue destruction, and up-regulation of this chemokine has
been linked with advanced lupus disease (18). A study of
MCP-1-/- MRL/lpr mice showed a
substantial delay in the induction of glomerulonephritis due to absence
of macrophages in kidney, although anti-DNA Ab production and
deposition in glomeruli remained intact (3).
IFN-+/- MRL/lpr mice constitute a
similar model, as they develop anti-DNA Abs, but not disease. Our
data show that kidneys from these mice are devoid of macrophages,
indicating that reduction in IFN- is crucial for progression of the
inflammatory process. Perivascular infiltrate was nevertheless present
in these mice as well as in IFN--/- kidneys,
indicating that kidney inflammation and lymphocyte migration are
initiated in the absence of immune deposits, perhaps as a result of an
overactive immune response in lpr mice. Because IFN- is
thought to induce increased MCP-1 production (26), we
examined whether the reduced macrophage migration into
IFN--/- and
IFN-+/- mouse kidneys was due to reduced
MCP-1 expression. MCP-1 was up-regulated in kidneys of both types of
mice. Conversely, MCP-1 appears not to interfere with IFN-
production, as suggested by recent data showing that absence of MCP-1
interferes with Th2, but not Th1 polarization, and that
MCP-1-/- mice produce normal IFN- levels
(27). Because MCP-1 was up-regulated equally in kidneys of
wt as well as IFN-+/- MRL/lpr
mice, which lack interstitial macrophage accumulation, we deduce that
IFN- contributes to macrophage accumulation by a mechanism
independent of MCP-1 expression.
The reduced ICAM-1 and VCAM-1 levels in
IFN-+/- mouse kidneys as compared with those
of wt MRL/lpr mice suggest that the mechanism for regulating
macrophage recruitment by IFN- is related to the adhesion process.
Although adhesion molecules may be up-regulated by chemokines
(28), this appears not to be the case for
IFN-+/- and
IFN--/- mice exhibiting high levels of
several chemokines in kidney. It thus appears that up-regulation
of these molecules depends on IFN- production by interstitial
macrophages. Experiments showing that transfer of
IFN-+/+ T cells was unable to restore
interstitial kidney inflammation or ICAM-1/VCAM-1 up-regulation
strengthen the view that macrophage-produced IFN- regulates the
expression of these adhesion molecules by tubular and mesangial cells.
On the basis of these data and the simultaneous up-regulation of
adhesion molecules and F4/80+ cell accumulation
in the kidney, it can be envisaged that macrophage-produced IFN-
enhances adhesion molecule up-regulation; this in turn consolidates
macrophage accumulation in the kidney interstitium. The ability of
macrophages to initiate the inflammatory cascade through their IFN-
secretory potential may be due to their migration into the
interstitium, whereas T cells, also an abundant IFN- source, remain
perivascular. Only macrophage-produced IFN- thus leads to adhesion
molecule up-regulation. Previous studies showed that, following IFN-
stimulation, tubular and mesangial cells up-regulate ICAM-1 and VCAM-1,
among other molecules (29, 30, 31). ICAM-1 is required for
leukocyte infiltration and ischemic renal injury (32).
Furthermore, ICAM-1 and VCAM-1 are up-regulated in the kidneys of lupus
model mice as well as in human disease (24). In
MRL/lpr mice, ICAM-1 deficiency has been implicated in
increased survival in two studies; in one, absence of pneumonia was
reported (33); in the second, a delay was shown in
glomerulonephritis onset (34). Nevertheless, absence of
ICAM-1 did not result in strong inhibition of interstitial inflammatory
cell recruitment, as is the case for IFN-+/-
mice. Although direct evidence is not available, the additive effect of
ICAM-1 and VCAM-1 down-regulation may impede macrophage accumulation in
kidney and effectively delay disease onset; up-regulation of other
molecules involved in macrophage chemoattraction or adhesion may also
be IFN--dependent.
To further understand the role of IFN- in macrophage attraction, we
sought to identify the source of IFN- required for kidney
inflammation. Our results show that in
IFN--/- MRL/lpr mice, adoptively
transferred IFN-+/+ macrophages migrate to
kidney peritubular and periglomerular zones, initiate adhesion molecule
up-regulation, and induce further macrophage migration. Although
IFN--producing macrophages are identified in the kidney
interstitium, we cannot exclude that IFN--/-
macrophages are also recruited in this organ. That macrophage
recruitment occurs in the absence of IC deposits indicates that the
inflammation process can be disengaged from IC deposition, which is
necessary for glomerulonephritis development. Macrophages accumulate in
the kidney interstitium, coinciding with the sites of adhesion molecule
up-regulation, suggesting that they are the primary cells involved in
inflammation. This result is further supported by data showing that
adoptive transfer of IFN-+/+ T cells to
IFN--/- mice does not reconstitute the
inflammatory process, as is the case of adoptively transferred
IFN-+/+ macrophages. The IFN- deficiency of
macrophages does not appear to affect the overall activation of T
cells. Indeed, in IFN--/- mice, reduced MHC
class II Ag expression by macrophages may cause reduced autoantibody
production, but does not affect hypergammaglobulinemia,
CD4+ and CD8+ T cell
proliferation (2), or the extent of perivascular
infiltration compared with IFN-+/+ mice. The
severe lung inflammation observed in the
IFN--/- mice adoptively transferred with T
cells supports the view that transferred T cells become activated.
Furthermore, CD3+ cell accumulation in the lung
suggests that although IFN- is an important inflammatory molecule,
distinct IFN--producing cells may exacerbate inflammation in
different organs.
Macrophage recruitment to the kidney is considered an important factor
in initiation of tissue destruction (3). In this study, we
show that impaired IFN- production inhibits macrophage recruitment
to the kidney in MRL/lpr mice, indicating that the level of
this cytokine is critical for disease onset and progression. In another
study, Tnf hemizygosity induces lupus development in the
otherwise lupus-resistant NZB mouse strain, presumably by influencing
early B cell autoimmune responses (35). The degree of
expression of these two cytokines thus appears critical for modulation
of lupus development.
Adoptive transfer of IFN-+/+ macrophages in
IFN-+/- mice, which have glomerular
anti-DNA deposits, reconstitutes glomerular disease; this indicates
that the degree of IFN- production by macrophages is a critical
factor in glomerulonephritis development. Based on these findings, we
propose a model describing the roles of IFN- and MCP-1 in macrophage
recruitment to the kidney of lupus-prone mice. MCP-1 may thus be
up-regulated in the kidney due to IC deposition or to an overactive
immune response. Elevated MCP-1 production provokes migration of
macrophages, whose ability to secrete large amounts of IFN- leads to
their adherence and residence in the kidney by up-regulating adhesion
molecules and perhaps other inflammatory factors. These destructive
cells then contribute to further inflammation and macrophage
recruitment. Under conditions of suboptimal IFN- production, as is
the case for IFN-+/- mice, MCP-1-attracted
macrophages do not initiate the adhesion process, probably due to
reduced IFN- secretion into the interstitial microenvironment; the
macrophages thus do not accumulate in the kidney interstitium. This
course of events concurs with the view that leukocyte migration is
initially regulated by selectin-dependent rolling, followed by
chemokine-mediated attraction; finally, the recruitment process is
completed by up-regulated adhesion (28).
Understanding the mechanism of the inflammation pathway in lupus
nephritis, as well as the molecules involved in this process, merits
study due to the potential for therapeutic intervention even after
autoantibodies are generated. This type of treatment may be useful in
the treatment of human SLE, because autoantibody production precedes
diagnosis. The results of this study suggest that targeting key
inflammatory factors such as MCP-1, in combination with adhesion
molecules or local IFN- production in the kidney, may constitute
promising strategies for treatment of autoimmune disease without
affecting critical IFN--mediated immune responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to: Dr. Dimitrios Balomenos, Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus de Cantoblanco, E-28049 Madrid, Spain. E-mail address: dbalomenos{at}cnb.uam.es
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; IC, immune complex; MCP, monocyte chemoattractant protein; wt, wild type; PO, peroxidase; PCNA, anti-proliferating nuclear Ag; BrdU, 5-bromo-2'-deoxyuridine; BUN, blood urea nitrogen; MIP, macrophage-inflammatory protein.
Received for publication August 13, 2001. Accepted for publication May 8, 2002.
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