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Renal Research Laboratory and
Department of Immunology, Fundación Jiménez Díaz, Autonoma University,
Hospital Clínico San Carlos, and
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Hospital Ramón y Cajal, Alcalá University, and
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Department of Biochemistry, Complutense University, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of FcR revealed that activation of resident cells
through FcR may be the initial mechanism of IC disease in the skin,
independent of complement activation (2, 3). These results
have been recently extended to murine models of IC-mediated nephritis
(4, 5). However, other authors reported that the
contribution of complement and FcR is diverse and that the pathogenesis
of the inflammatory response may be tissue and strain specific
(6). Interaction of IC with resident glomerular mesangial cells (MC) triggers a cascade of events characterized by mononuclear cell recruitment, cell proliferation, and matrix protein accumulation (1). Among the different mediators released during renal injury, chemokines, a superfamily of small proinflammatory peptides, are important in the recruitment and activation of leukocytes (7). In general, the C-X-C chemokines, such as IL-8 and IFN-inducible protein-10 (IP-10) act predominantly on neutrophils, whereas the C-C family, such as monocyte chemoattractant protein-1 (MCP-1) acts on lymphocytes, monocytes, mast cells, and eosinophils (8). A number of studies has demonstrated an overexpression of MCP-1, IP-10, and IL-8 [or the functional homologue of IL-8 in rat, cytokine-induced neutrophil chemoattractant (CINC)] by inflammatory and resident cells in several experimental models of IC disease, including glomerulonephritis (9, 10, 11, 12, 13).
Platelet-derived growth factor (PDGF) and TGF-ß are probably the most important mediators involved in MC proliferation and extracellular matrix synthesis associated with glomerulonephritis (14, 15). In vitro, PDGF is mitogenic and chemoattractant for MC, induces cell contraction, stimulates synthesis of cytokines, and may regulate matrix production both directly and through other growth factors (14). TGF-ß stimulates the synthesis of fibronectin, proteoglycans, and collagens in several cell types, including MC (16, 17, 18).
Transcription factors, such as NF-
B and AP-1, regulate the gene
transcription of several cytokines, complement components, chemotactic
proteins, adhesion molecules, and matrix proteins involved in
inflammation, immunological responses, cell differentiation, and growth
control (17, 19, 20, 21, 22). NF-B is a dimer of p50 and p65
subunits (19), and AP-1 is composed of homodimers and
heterodimers of Jun and Fos proteins (20). The activation
of these transcription factors during glomerular injury has been
scarcely studied (23, 24).
In recent years, several groups have described the presence of FcR for IgG and IgA in rat and human MC (25, 26, 27) and several intracellular signals induced through FcR (28, 29, 30). The stimulation of resident MC with IC elicits synthesis of proinflammatory and profibrogenic cytokines and complement components, as well as mesangial proliferation and matrix production (18, 30, 31, 32, 33, 34). In vitro, these cell responses were markedly decreased by the presence of Fc fragments of Igs in the culture medium (18, 28, 30, 33). Therefore, we speculated that the in vivo administration of Fc fragments could attenuate the inflammatory response and other biological changes elicited by IC.
In a model of IC nephritis in rats, characterized by marked glomerular
immune deposits, cell proliferation, matrix accumulation, and intense
proteinuria (35, 36), we have studied the effect of
systemic injection of highly purified Fc fragments of IgG. In addition,
we analyzed whether the therapeutic effect of Fc fragment
administration may involve a reduced renal production of complement
components (C3), chemokines (MCP-1, IP-10, and CINC-1), and growth
factors (PDGF and TGF-ß), as well as the attenuation of transcription
factor activation (NF-B and AP-1) that regulates their expression.
The beneficial effect observed in animals treated with Fc fragments
paralleled the effects observed in vitro, suggesting that FcR could be
an important target in the therapeutic approach to severe immune
nephritis. In addition, our data could be extended to other IC-mediated
diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fc fragments from rabbit IgG were obtained by digestion with activated papain (Sigma, St. Louis, MO), which does not need reducing agents to assure the integrity of the inter-H-H disulfide bridge (37). The Fc fragment (molecular mass of about 60 kDa) was purified by affinity chromatography on a protein A-Sepharose column and gel filtration on a Superdex 75 column in PBS (Pharmacia Biotech, Uppsala, Sweden) and concentrated to 2 mg/ml, and did not contain any detectable Fab fragment, IgG, or other Igs. After analysis on an Applied Biosystem 470-A gas phase sequencer (Applied Biosystems, Foster City, CA), an N-terminal sequence of Cys-Pro-Pro-Pro-Glu-Leu-Leu-Gly-Gly-Pro was obtained, indicating that the purified Fc fragments began at Cys 229 (this residue forms the inter-H-H bridge). Purified Fc fragments and BSA (Sigma) were radiolabeled with 1 mCi Na125I (Amersham, Buckinghamshire, U.K.) using the chloramine-T method (sp. act., 0.1 mCi/mg). Fc fragments were biotinylated by incubation with sulfosuccinimidyl-6-(biotinamido) hexanoate (Pierce, Rockford, IL).
Experimental design
Preliminary studies were performed in healthy female Wistar rats to establish the optimal conditions of the Fc fragment treatment. Animals were injected i.p. with 1 mg of 125I-labeled Fc fragments, and at different time points the organ distribution and pharmacokinetics were determined in a gamma counter. As control, radiolabeled albumin was injected into a parallel group of animals to determine the nonspecific trapping of radioactivity. The specific distribution of Fc fragments was expressed as percentage vs injected dose, and the background in the different organs (albumin distribution) was corrected at each time point. In other experiments, rats were injected with 1 mg of biotinylated or unlabeled Fc fragments or with the same volume of PBS containing the reactive for biotinylation (sulfosuccinimidyl-6-(biotinamido) hexanoate). The localization of biotin in renal sections was analyzed by staining with avidin-biotinylated HRP (Dako, Glostrup, Denmark).
The experimental model of IC nephritis was performed in 200-g female Wistar rats according to a previously described protocol (35, 36). Briefly, rats received an initial s.c. injection of 5 mg OVA (Sigma) in CFA (Difco, Detroit, MI), and, after 3 wk, the same dose was given in IFA. One week later, daily i.p. administration of 10 mg OVA was started. When proteinuria reached 20–50 mg/24 h, animals were randomly distributed into two groups: 1, animals with a daily i.p. injection of 1 mg Fc fragments (Fc-treated group, n = 10), and 2, animals with spontaneous development of nephritis (untreated group, n = 9), receiving PBS, the vehicle of Fc fragments. Parallel control groups of healthy rats injected with Fc fragments or vehicle (n = 6) were also studied. Two weeks later, animals were sacrificed, blood was collected, and kidneys were removed. Proteinuria in daily collected urine samples was measured using the sulfosalicylic acid method. Serum levels of creatinine, albumin, and cholesterol were determined by standard methods.
Complement measurements
Serum levels of the C3 component of complement were measured by radial immunodiffusion with sheep anti-rat C3 (The Binding Site, Birmingham, U.K.), and the data were expressed as percentage vs a standard rat serum pool. The hemolytic activity of complement in sera was analyzed using sheep erythrocytes sensitized with anti-sheep hemolysin (Sigma), and the hemolytic titer was expressed in total complement hemolytic activity (CH50) U/ml (37). In some cases the CH50 titer of a control rat serum was determined in the presence of increasing amounts of IgG IC or Fc fragments. These IC were obtained after incubation of rabbit anti-OVA IgG (1 mg) with OVA (50 µg) for 1 h at 37°C (38). The consumption of complement was expressed as percentage of inhibition vs basal (hemolysis observed in the presence of rat serum without IC or Fc fragments). The binding of human C3 to IC was performed as described (38, 39). Briefly, normal human serum containing iodo[1-14C]acetamide was incubated at 37°C with IgG IC (100 µg) in the presence or absence of Fc fragments (100 µg), and covalent complexes were analyzed by SDS-PAGE and fluorography. The intensity of the bands corresponding to C3b-IgG complexes was quantified by densitometry.
Histopathological studies
Renal cortex was snap frozen in liquid nitrogen and stored until analysis. Additional slices of cortex were immersed in 10% neutral-buffered Formalin and embedded in paraffin. Light microscopy was performed on paraffin sections (5-µm thick) stained with hematoxylin-eosin, Masson’s trichrome, and silver methenamine. The distribution of IgG, Fc fragments, C3, and matrix proteins was analyzed in frozen sections by immunofluorescence with rabbit anti-rat IgG (Sigma), anti-rabbit IgG (Sigma), sheep anti-rat C3, rabbit anti-rat fibronectin (36), and goat anti-rat type IV collagen (Southern Biotechnology, Birmingham, U.K.) Abs, respectively. Deposits of C1q were analyzed by immunoperoxidase with rabbit anti-human C1q Ab produced in our laboratory (40). The specificity of this Ab was determined by Western blot using human and rat sera in comparison with purified human C1q. The quantification of infiltrating and proliferating cells was performed on frozen tissue specimens by an indirect immunoperoxidase technique with the following Abs: OX1 (pan-leukocyte CD45 Ag), OX19 (CD5 Ag), OX6 (rat MHC class II Ag, RT1-B), ED1 (rat monocyte/macrophages) (Serotec, Oxford, U.K.), and proliferating cell nuclear Ag (PCNA; Dako). Immunohistochemistry for chemokines was analyzed in paraffin-embedded renal tissues by incubation with goat anti-human MCP-1 (50 µg/ml; Immugenex, Los Angeles, CA), goat anti-mouse IP-10 (10 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), and goat anti-rat CINC-1 (10 µg/ml; R&D System, Oxon, U.K.). After treating with peroxidase-conjugated secondary Abs, the samples were developed with 3,3'-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Negative controls were run in parallel with the omission of primary Abs.
Analysis of mRNA expression
Total RNA from rat renal cortex pieces was obtained by the acid
guanidine-thiocyanate-phenol-chloroform method, and the mRNA expression
of rat C3, TGF-ß, and PDGF A chain was analyzed by RT-PCR using the
following primers (Genosys, Cambridge, U.K.): C3 (693 bp): 5' primer,
5'-GGA AGT GTT GTG AGG ATG GCA-3'; 3' primer, 5'-CTG ATG AAG TGG TTG
AAG ACG G-3'; TGF-ß (298 bp): 5'-primer, 5'-C TTC AGC TCC ACA GAG AAG
AAC TGC-3'; 3' primer, 5'-CAC GAT CAT GTT GGA CAA CTG CTC C-3'; and
PDGF A chain (312 bp): 5' primer, 5'-GAG ATA CCC CGG GAG TTG AT-3'; 3'
primer, 5'-CTT CAC CTC CAC ACA TGG G-3'. A total of 1 µg of RNA was
reverse transcribed to single-stranded cDNA, and then a PCR containing
20 pmol of primers, 0.5 µCi [
-32P]dCTP (3000Ci/mmol;
Amersham) and 3 U Taq DNA polymerase was conducted with
annealing temperatures of 56°C (C3), 60°C (TGF-ß), or 57°C
(PDGF). The expression of G3PDH was used as internal control.
For in situ hybridization, riboprobes were generated by ligation of the PCR product into a PCR 3 vector (Invitrogen, San Diego, CA), and labeled anti-sense and sense cRNA probes were generated using T7 or SP6 polymerases and digoxigenin-labeled UTP as substrate (Boehringer Mannheim, Mannheim, Germany). Paraffin-embedded tissue sections were fixed in 1.5% paraformaldehyde-1.5% glutaraldehyde and treated with 5 mM levamisole, 0.2 N HCl, and 25 µg/ml proteinase K. Hybridization was conducted at 42°C with 0.4 ng/µl denatured digoxigenin-labeled riboprobes. Sections were washed and incubated with alkaline phosphatase conjugated anti-digoxigenin Ab (Boehringer Mannheim). Colorimetric detection of mRNA was performed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate. Negative controls consisted of matched serial sections hybridized without probe, with sense probe, or pretreated with 25 µg/ml RNase A.
Activity of transcription factors
Nuclear protein extraction from frozen renal tissue samples and
EMSA were performed as described (24, 30). NF-B and
AP-1 consensus oligonucleotides were radiolabeled with
[-32P]ATP (3000 Ci/mmol; Amersham). A total
of 10 µg of nuclear protein was incubated in buffer containing 50
µg/ml poly(dI-dC) (Pharmacia), and 0.035 pmol of the labeled probe.
The reactions were analyzed on a 4% nondenaturing polyacrylamide gel
and autoradiographed. Specificity of the binding reaction was confirmed
using a 100-fold excess of unlabeled specific oligonucleotides.
Identification of nuclear proteins bound to the oligonucleotides was
performed by using Abs against the p50, p65, and c-Rel subunits of
NF-B or the c-Jun and c-Fos subunits of AP-1 (Santa Cruz
Biotechnology).
The Southwestern histochemistry was developed to detect the in situ
distribution and DNA-binding activity of transcription factors
(41). NF-B and AP-1 consensus oligonucleotides were
digoxigenin labeled with a 3' terminal transferase (Boehringer
Mannheim). Frozen and paraffin-embedded tissue sections were fixed in
0.5% paraformaldehyde and incubated with 0.1 mg/ml DNase I. The
DNA-binding reaction was performed by incubation with 10 pmol of the
labeled DNA probe in buffer containing 0.25% BSA and 0.5 µg/ml
poly(dI-dC). The sections were then incubated with alkaline
phosphatase-conjugated anti-digoxigenin Ab, and colorimetric
detection was performed as described. Preparations without probe were
used as negative controls, and mutant-labeled probe and excess of
unlabeled probe were used to test the specificity of the technique.
Evaluation of tissue staining and statistical analysis
In glomerular, tubular, and interstitial areas, around 20 fields from each animal were examined without any previous knowledge about the experimental design. The stainings in the different renal structures were graded semiquantitatively on a scale from 0 to 3 (0, negative; 1, weak; 2, moderate; and 3, strong staining), and data were expressed as means ± SEM. Statistical analysis was performed by ANOVA, Student-Newman-Keuls, or Tukey-Kramer tests. Differences were considered to be significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Radiolabeled Fc fragments of IgG (1 mg) were i.p. injected into healthy control rats, and, after 3, 6, 9, and 24 h, animals were killed and the radioactivity in selected organs (spleen, heart, lung, liver, and kidney) was measured. The data of Fc fragment distribution were corrected for background, considered as the unspecific trapping of a non-Fc-related protein (radiolabeled albumin) injected into a parallel group of animals. The analysis of the specific distribution revealed that the maximal accumulation of Fc fragments was detected in liver and kidneys, peaking at 3 h after injection (17 and 18% vs injected dose, respectively) and decreased after 24 h (4 and 5%, respectively).
The biodistribution of Fc fragments in the kidney was analyzed in
control rats injected with biotinylated Fc fragments. After staining
with avidin-biotinylated peroxidase, positivity for biotin was found in
the glomeruli, with a diffuse distribution, whereas no staining was
observed in rats injected with unlabeled Fc fragments or PBS containing
the biotinylation reactive (data not shown). Additionally,
immunofluorescence studies with a specific Ab against rabbit IgG (no
cross-reactivity with other species) showed the presence of Fc
fragments in the glomeruli of control rats treated for 2 wk with 1 mg
of Fc fragments (Fig. 1
A). For
all of these reasons, we thought it appropriate to perform the
treatment with a daily i.p. injection of 1 mg of Fc fragments for
2 wk.
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In the model of proliferative nephritis employed, proteinuria
rises above normal values around the 9th wk after the first Ag
injection (35, 36). When proteinuria reached 20–50 mg/24
h, animals were randomly assigned to receive either 1 mg/day of Fc
fragments (Fc-treated group) or vehicle (untreated group) for 2 wk. At
the start of therapy, proteinuria was 31 ± 4 and 33 ± 7
mg/24 h, respectively (NS). As shown in Fig. 1
, urinary protein
excretion in untreated animals increased over the 2 wk of study,
progressing to a full-blown nephrotic syndrome. The administration of
Fc fragments prevented the development of intense proteinuria (86%
reduction in relation to the untreated animals).
Untreated animals developed renal dysfunction, as evidenced by reduced
creatinine clearance, hypoproteinemia, and hypercholesterolemia (Table I). Animals treated with Fc fragments
showed a preservation of the creatinine clearance and near
normalization of serum albumin and cholesterol levels. Healthy control
rats receiving Fc fragments or vehicle displayed serum and renal
parameters in the normal range (Table I).
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This model is characterized by the presence of a large amount of
immune deposits in the mesangium and glomerular capillary wall
(36). Immunofluorescence analysis with anti-rat IgG
revealed no significant differences in the deposition of IgG among
glomeruli from untreated and Fc-treated rats (3 ± 0 vs 2.8
± 0.1; semiquantitative score, NS) (Fig. 2, E and F).
No staining for rat IgG was detected in control animals treated with
vehicle (data not shown) or Fc fragments (Fig. 2D). The
renal distribution of Fc fragments after injection was analyzed by
immunofluorescence with anti-rabbit IgG. As indicated in Fig. 2, A and C, Fc fragment staining was similar both in
control and nephritic animals treated with Fc fragments for 2 wk,
whereas no staining was observed in nephritic rats injected with
vehicle (Fig. 2B).
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K) and C3 (Fig. 2H), were present in the kidney
of nephritic animals, mainly in the glomerular area and in some tubuli.
The semiquantitative analysis revealed no significant differences in
the localization and intensity of C1q between Fc-treated and untreated
nephritic rats (2.5 ± 0.2 vs 2.7 ± 0.1, NS) (Fig. 2L). In contrast, a decrease in the glomerular staining of
C3 was noted after Fc fragment administration (0.2 ± 0.1 vs
2.5 ± 0.3, p < 0.05) (Fig. 2I).
Control rats injected for 2 wk with Fc fragments showed a very low
staining for both complement components (Fig. 2, G and
J). Analysis of complement
Since recent data suggest that locally generated complement could
be important in tissue injury (42, 43, 44), we studied the
gene expression of C3 in renal tissues of rats. Fig. 3, A–C, is a representative
example of in situ hybridization for C3 mRNA. Few glomerular and
tubular cells positive for C3 mRNA were observed in the kidney of
healthy control rats injected with Fc fragments (Fig. 3A) or
vehicle (data not shown). In diseased kidney, C3 mRNA expression was
increased in the glomerulus (with positivity found mainly in the
mesangial area, and in some epithelial cells and Bowman’s capsule
cells), tubular epithelial cells, and interstitial infiltrating cells
(Fig. 3B). In contrast, the administration of Fc fragments
diminished this local expression of C3, since a low amount of C3
transcripts was detected in rats with ongoing nephritis treated with Fc
fragments for 2 wk (Fig. 3C; semiquantitative glomerular
score, 0.4 ± 0.2 vs 2.8 ± 0.2, p < 0.01).
The hybridization signal was highly specific, as assessed by several
negative controls, such as hybridization with the sense probe or
pretreatment with RNase A (data not shown). These results were
confirmed by RT-PCR analysis (Fig. 3D). After densitometry
and correction for the G3PDH expression, the 3-fold increase in the
mRNA expression of C3 observed in the kidneys of rats with nephritis
was reduced to control levels after Fc fragment administration (Fig. 3D).
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, C3 and CH50 values were
unmodified in healthy and nephritic animals injected for 2 wk with Fc
fragments or vehicle, indicating that the treatment did not affect
systemic complement. In addition, several in vitro experiments were
performed to assess whether Fc fragment preparation could modify the
activation of the complement system. Sensitized sheep erythrocytes were
incubated with IC or Fc fragments in the presence of control rat serum
as complement source. Hemolysis of sensitized erythrocytes (CH50 value)
was dramatically decreased by incubation with increasing concentrations
of IC, reaching a plateau of 100 µg/ml (Fig. 4A). In contrast, a very low
consumption of complement was observed after incubation with Fc
fragments (Fig. 4A). The maximal reduction of hemolysis was
about 17% (300 µg/ml, Fig. 4A) even when very high
concentrations of Fc fragments (0.5 and 1 mg/ml) were employed (data
not shown). We also studied the effect of Fc fragments on the C3b
binding to preformed IgG IC. Upon interaction of serum C3 with IC,
C3b-C3b-IgG covalent complexes are immediately formed. These C3b-C3b
dimers constitute the core for the assembly of C3/C5 convertase on the
IC, which subsequently are converted to iC3b-iC3b-IgG by the complement
regulators (38). These complexes were detected on SDS-PAGE
and fluorography by two bands (of high molecular mass) with molecular
composition C365- C343 (band A) and C365-H of the IgG (band
B), which correspond to C3b-C3b and C3b-IgG covalent interaction,
respectively, and that identify opsonized IC (37, 38, 39)
(Fig. 4B, lane 1). The molecular mass observed
for bands A and B in a highly cross-linked gel is different from that
theoretically calculated, indicating the influence of the shape on the
apparent molecular mass, as previously reported (39). In
the presence of Fc fragments, the C3b covalent binding to IC was weakly
enhanced (Fig. 4B, lanes 2 and 3).
After densitometric analysis, we found that the intensity of bands A
and B increased 128 ± 3 and 116 ± 4 (percentage vs control
IC), respectively. However, we did not detect new bands corresponding
to C3b-Fc covalent complexes. These results suggest that the Fc
fragment was not sequestering C3.
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The morphological aspects of the glomerular lesions in this model
have been described previously (35, 36). Quantification of
renal damage is indicated in Table II.
Animals with nephritis showed glomerular hypercellularity,
mesangial expansion, severe necrotizing lesions, and inflammatory cell
infiltration. In contrast, the Fc-treated animals showed a
significant decrease in the structural glomerular damage, with discrete
evidence of mesangial hypercellularity and matrix expansion. The
tubulointerstitial lesions were also significantly reduced (Table II).
Glomerular and tubular structures were well preserved in the kidney of
control rats injected with Fc fragments or vehicle.
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). In contrast, Fc fragment treatment was associated with a
marked reduction in the number of proliferating cells. Fc-treated rats
had also less glomerular infiltration of total leukocytes, T
lymphocytes, and ED1+ monocyte/macrophages than
rats with untreated nephritis (Table II). Detection of chemokines in renal tissues
The presence of chemokines involved in the leukocyte recruitment
was determined using an indirect immunoperoxidase technique. Control
rats injected with Fc fragments or vehicle presented a low amount of
MCP-1 in the mesangium, whereas staining for IP-10 and CINC-1 was
negative (Fig. 5A,
D, and G). Induction of the IC nephritis caused a
marked increase in renal chemokine production at the end of the study.
Untreated nephritic rats presented a strong staining for MCP-1, IP-10,
and CINC-1 in glomerular and tubular cells, as well as in inflammatory
cells infiltrating the interstitium (Fig. 5, B,
E, and H). The increase in chemokine production
by both infiltrating and intrinsic kidney cells was largely prevented
by Fc fragment treatment, with a slight positivity in glomeruli and
some tubular cells (Fig. 5, C, F, and
I). Semiquantitative evaluation revealed a significant
decrease in the glomerular chemokine production in comparison with the
untreated nephritic animals (1.5 ± 0.1 vs 3 ± 0,
p < 0.01 for MCP-1; 0.5 ± 0.2 vs 2.5 ±
0.2, p < 0.001 for IP-10; 0.7 ± 0.2 vs 2.8
± 0.1, p < 0.001 for CINC-1).
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In normal kidneys, fibronectin is present in the mesangium and
along the glomerular basement membrane (data not shown).
Immunofluorescence studies showed that fibronectin increased in the
mesangium, capillary wall, tubular basement membrane, and interstitium
of nephritic animals (Fig. 6A). Positive immunostaining
for type IV collagen was seen along the glomerular basement membrane
and in the mesangial matrix and tubules (Fig. 6C).
Administration of Fc fragments diminished the glomerular deposition of
both matrix components (1.6 ± 0.3 vs 2.9 ± 0.1,
p < 0.05 for fibronectin; 1.2 ± 0.2 vs 2.8
± 0.2, p < 0.01 for type IV collagen) (Fig. 6, B and D).
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The cellular distribution of these growth factors in the kidneys
of animals from different groups was investigated by in situ
hybridization. Kidneys from normal rats injected with Fc fragments as
control expressed very low levels of PDGF and TGF-ß mRNA (Fig. 7, A and D), which
increased dramatically in animals with nephritis. Positivity for PDGF
in nephritic animals was found mainly in the mesangium and in some
tubular epithelial cells, particularly in distal tubules (Fig. 7B). Expression of TGF-ß was seen in the mesangial area,
the glomerular endothelial cells, and in infiltrating cells around the
tubules (Fig. 7E). Animals treated with Fc fragments showed
a minimal expression of both growth factors when compared with
untreated rats (0.4 ± 0.2 vs 2.6 ± 0.2, p
< 0.001 for PDGF; 0.5 ± 0 vs 2.7 ± 0.3, p
< 0.01 for TGF-ß) (Fig. 7, C and F).
Hybridization with the sense probe was performed as negative control of
the technique (data not shown). The in situ hybridization results were
confirmed by RT-PCR analysis. As indicated in Fig. 8, the up-regulation of both PDGF and
TGF-ß mRNA expression observed in the kidneys of rats with nephritis
(2- and 2.5-fold, respectively) was normalized to basal levels in
Fc-treated animals.
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The cortical transcription factor activity was analyzed in nuclear
proteins by EMSA. Extracts from nephritic rats showed a marked increase
in the intensity of the NF-B and AP-1 bands, indicating
transcription factor activation (Fig. 9).
The activity of both transcription factors was largely prevented by Fc
fragment administration since the intensity of the bands was
dramatically decreased (Fig. 9). The specificity of transcription
factor activity was verified by competition experiments with an excess
of unlabeled probes. Moreover, the appearance of supershifted bands
and/or diminution of the band intensity was observed after incubation
of nuclear extracts with Abs against p50, p65, c-Jun, and c-Fos,
suggesting that activated NF-B and AP-1 complexes are constituted by
p50/p65 and c-Jun/c-Fos proteins, respectively (Fig. 9).
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B and AP-1 was observed in renal tissues
from healthy control rats receiving Fc fragments (Fig. 10A and E) or
vehicle (data not shown), whereas the number of positive cells was very
increased in nephritic rats (Fig. 10, B and F).
Transcription factors were widely distributed in glomerulus and
tubulointerstitium, with positive nuclear staining in MC, glomerular
epithelial cells, some tubular epithelial cells, and mononuclear
infiltrating cells. In contrast, a small number of cells was stained in
the group of rats treated with Fc fragments (Fig. 10, C and
G) (semiquantitative score, 0.2 ± 0.1 vs 2.6 ±
0.1 for NF-B; 0.2 ± 0.1 vs 2.5 ± 0.3 for AP-1). As
negative controls to determine the specificity of the DNA-binding
reaction, sections were incubated without the probe (data not shown),
with the mutant-labeled probe (Fig. 10D), or with an
excess of consensus unlabeled probe (Fig. 10H).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The beneficial effects of Igs administration to patients with immune diseases have been extensively described, but the mechanisms are still not well defined (45, 46, 47, 48). Among them, FcR blockade, anti-idiotypic Ab interactions, prevention of the complement activation, and modulation of immune function were considered (45). A number of studies has also reported that the administration of Abs against some chemokines, cytokines, and growth factors exerts a beneficial effect on the course of various experimental glomerulonephritis (9, 10, 11, 14, 16). However, given the complex and redundant cytokine network, it is relatively improbable that the pharmacological modulation of one single mediator could have a major role in the treatment of human glomerulonephritis. Since the interaction of deposited and/or locally formed IC with resident glomerular cells bearing FcR (MC and macrophages) is one of the first steps in the pathogenesis of immune renal injury (1), our therapeutic approach of Fc fragment injection could be more rational. In contrast to these published studies in which treatment with Igs or specific Abs was mostly prophylactic (9, 10, 11, 16, 45, 47), we administered the IgG Fc fragments when the animals had a certain kidney damage with a proteinuria two to five times higher than that of controls. This is the first report on the therapeutic approach of Fc fragment administration in experimental nephritis, but the results are in concordance with two recent studies performed in acute immune thrombocytopenic purpura (49) and xenogenic hyperacute rejection (50).
The Fc fragment preparations employed in this study were highly purified, and the tissue distribution clearly suggests that they were principally taken up by the kidney and liver and persisted for at least 24 h. The immunostaining revealed the presence of Fc fragments in the glomeruli of healthy rats several hours after injection. Additionally, the same pattern of mesangial localization, both in healthy and nephritic rats treated with Fc fragments, was observed at the end of the study, suggesting that the Fc fragment distribution into the kidney was not affected by the disease. The i.p. route was chosen because the IgG plasma levels in rodents were similar when IgG was administered i.p. or i.v. (47). The amount injected (1 mg/day) was well tolerated, with no adverse reactions, in agreement with that observed in humans and animals employing higher doses of either IgG or Fc fragments (46, 47, 48, 49, 50, 51). Thus, we consider that administration of Fc fragments could be more effective than intact IgG with additional advantages. The lower m.w. of Fc fragments would allow a better localization in the injured kidney. In addition, Fc fragments contain all of the structural elements necessary to induce the effector functions of IgG. The Fc portion of IgG could also be easily obtained by gene technology avoiding the large pool of blood donors needed for the complete IgG molecule, thus providing a cheaper and safer treatment.
Since both deposited IgG IC and injected Fc fragments colocalize in the mesangium of Fc-treated nephritic animals, we postulate that the blockade of FcR could be the underlying mechanism of the beneficial effect of Fc fragments. As a consequence, activation of renal cells by IC and local production of inflammatory proteins involved in leukocyte recruitment could not occur. In this work, we show that the administration of Fc fragments markedly reduces the number of proliferating and infiltrating cells and the extracellular matrix synthesis in glomerular and tubulointerstitial areas. Among the molecules involved in these processes, chemokines and growth factors seem to play a major role. Chemokines are known to be secreted by infiltrating mononuclear and resident renal cells in response to various stimuli, including IC (30, 31, 32). The stimulation of cultured MC with IC via FcR also triggers the formation of extracellular matrix proteins, a process partially mediated by the autocrine synthesis of TGF-ß (18). The increased expression of chemokines and growth factors was described in several models of renal injury (9, 10, 11, 12, 15, 16, 24, 36), and the glomerular lesions were improved by administration of neutralizing Abs (9, 10, 11, 14, 16). In our experimental model, we observed that in temporal association with leukocyte infiltration and glomerular damage, there was an up-regulation of MCP-1, IP-10, and CINC-1, largely prevented by treatment with Fc fragments both at the glomerular and tubulointerstitial levels. Additionally, the renal localization of PDGF and TGF-ß was analyzed by in situ hybridization, observing a reduced renal mRNA expression of both growth factors in parallel with a diminution in MC proliferation and extracellular matrix accumulation.
The Fc fragments might also attenuate IC-mediated renal injury through additional mechanisms. First, the Fc fragment preparation can decrease the formation, clearance, and deposition of IC into the glomerulus. This is unlikely because no changes in the localization and intensity of glomerular IgG were observed after the treatment. Second, the Fc fragments could have a systemic action on the serum complement levels. However, there was no evidence of systemic complement activation and depletion, since C3 and CH50 levels were unmodified by the treatment. These data are supported by the in vitro studies with Fc fragments, which revealed a very weak complement consumption. Third, Fc fragments can interfere in the kidney by preventing the local synthesis and/or activation of complement. Our finding of a decrease in the glomerular staining of C3 (an essential component of both classical and alternative pathways of complement) after Fc fragment injection supports this mechanism.
It is well known that very high doses of IgG inhibit the deposition of
C3b onto target surfaces diverting nascent C3b from tissue-bound IC to
fluid phase IgG, forming C3b-IgG covalent complexes (51).
However, in our system, the Fc fragments themselves (at a similar
concentration as IgG IC) were not acceptors of C3b, and we did not
detect the presence of C3b-Fc covalent complexes. Interestingly, the
increased renal C3 mRNA expression observed in nephritic animals was
reversed to control levels by the treatment, suggesting that Fc
fragments are blocking the renal synthesis of C3. The importance of the
local C3 synthesis has recently been reported in C3-deficient mice
(44). In the kidney, several authors have described an
increased renal expression of complement components in
cytokine-stimulated cultured renal cells (52) and in
glomerulonephritis (42, 43), and proposed that the
activated, locally synthesized C3, may change the size and/or charge
barrier of the glomerular basement membrane, causing proteinuria
(42). Moreover, IC enhanced the synthesis of C3 by
cultured human MC via FcR (34). In a murine model of IC
nephritis, a reduction of the disease was observed in mice with a
genetic deficiency in the subunit of FcR, but not after
decomplementation with cobra venom (5), indicating that
complement activation induced by mesangial IC deposition contributes to
the pathogenesis of immune renal injury but could not be sufficient to
initiate the inflammatory response. Thus, our data suggest that one of
the mechanisms of the beneficial effect of Fc fragments seems to be the
inhibition of renal complement expression, without serum complement
modification. This could be a potential advantage for their therapeutic
use in humans.
Nuclear translocation of active transcription factors and binding to
specific DNA sequences of responsive genes can lead to their enhanced
transcription and subsequent protein synthesis (19, 20).
Transcription factor activity has recently been found in experimental
nephritis and in cultured MC stimulated with IC (23, 24, 29, 30). In this work, we analyze the activation and localization of
NF-B and AP-1, transcription factors involved in the gene regulation
of complement components, chemokines, growth factors, and matrix
components (17, 21, 22). The intense nuclear staining
observed in cells from glomeruli and tubulointerstitium of animals with
untreated nephritis virtually disappeared in the Fc-treated group.
Therefore, our data suggest that Fc fragments may prevent the
stimulation via FcR and thus inhibit the intracellular signaling that
leads to the activation of a variety of nuclear transcription factors.
Consequently, the transcription of a large number of genes involved in
inflammation and in tissue injury was prevented.
On the whole, we have demonstrated that the administration of Fc fragments to animals with IC nephritis prevents the appearance of severe nephrotic syndrome, renal failure, and glomerular and tubulointerstitial lesions by interfering with the initial steps of renal injury. These data suggest that the FcR could be an important target in the therapeutic approach to IC diseases. Prospective control trials are needed to assess whether Fc fragments administration, alone or in combination with immunosuppressive drugs, may play a beneficial role in the treatment of severe glomerulonephritis in humans.
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Acknowledgments |
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Footnotes |
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2 C.G-G. and N.D. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Jesús Egido, Renal and Vascular Research Laboratory, Fundación Jiménez Díaz, Avda Reyes Católicos 2, 28040 Madrid, Spain. E-mail address:
4 Abbreviations used in this paper: IC, immune complexes; FcR, Fc receptors; MC, mesangial cells; MCP-1, monocyte chemoattractant protein-1; IP-10, IFN-inducible protein-10; CINC-1, cytokine-induced neutrophil chemoattractant-1; PDGF, platelet-derived growth factor; PCNA, proliferating cell nuclear Ag; CH50, total complement hemolytic activity.
Received for publication January 22, 1999. Accepted for publication November 4, 1999.
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) was
expressed in mg/24 h as mean ± SEM. *, p <
0.05 vs untreated nephritic rats.
).
Consumption of complement was expressed as percentage of inhibition
respect to hemolysis in control conditions (CH50 value of control rat
serum). Results are means ± SEM of three different experiments
performed in duplicate. B, The covalent interaction of serum
C3 with IC was analyzed by SDS-PAGE after incubation of IgG IC (100
µg) in the absence (lane 1) or presence (lanes
2 and 3) of Fc fragments (100 µg). Two bands
characterize those IC which have activated complement, bound C3b (band
B), and are able to generate the C3/C5 convertase (band A).
Representative of two different experiments.
