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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3467-3472
IMMUNOBIOLOGY
Heavy and light chain primary structures control IgG3
nephritogenicity in an experimental model for cryocrystalglobulinemia
Jens-Uwe Rengers,
Guy Touchard,
Catherine Decourt,
Sophie Deret,
Hartmut Michel, and
Michel Cogné
From the Max-Planck-Institute for Biophysics, Frankfurt, Germany;
the Laboratory of Immunology, CNRS EP118, University Hospital, Limoges,
France; the Nephrology Department, University Hospital, Poitiers,
France; and the Clinical Immunology Department, INSERM U25, Paris,
France.
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Abstract |
Crystal formation by monoclonal immunoglobulins is a well-known but
rare complication of B-cell neoplasia. We have designed an in vivo
model of cryocrystalglobulinemia by grafting to mice hybridoma clones
producing a pathogenic monoclonal immunogloblulin (Ig) G3 . One
clone, 8A4, secreted a singular IgG3 that formed crystals both in the
proliferating plasma cells and as mesangial and subendothelial deposits
in the kidney glomeruli. Morphologic analysis of kidneys revealed
neutrophil infiltration and endocapillary hyperplasia, while the
morphology of deposits was reminiscent of those in
cryocrystalglobulinemia patients. A variant clone that only differed
from 8A4 by a 3-amino acid deletion in the V CDR1
increased its secretion level by 7-fold and produced an abundant bona
fide serum monoclonal cryoglobulin in mice, without crystal formation
within tumoral cells; it yielded no subendothelial deposits but only
amorphous precipitates in capillary lumens of kidney glomeruli,
reminiscent of those seen in the human hyperviscosity syndrome, without
other glomerular lesions. A limited variation in the V
domain thus proved able to increase secretion, to abrogate
crystallization, and to modify patterns of glomerular lesions and
deposits. Both the crystallizing and noncrystallizing IgG3 sequences
were related to previously reported murine cryoglobulins, all including
a  3 chain and canonical VH sequences. Two additional variants of 8A4
with identical VH and VL domains but having switched to IgG1 also lost
crystal formation, further showing this feature of 8A4 to result from a
unique 3-dimensional conformation of the complete immunoglobulin,
relying on V and C domain primary structures of both chains.
(Blood. 2000;95:3467-3472)
© 2000 by The American Society of Hematology.
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Introduction |
Cryocrystalglobulinemia is characterized by
crystallization of a monoclonal immunoglobulin (Ig) within plasma cells
and as crystal deposits affecting various tissues, especially the
kidneys. It occurs in humans as a rare complication of B-cell
immunoproliferative disorders, first described by Von Bonsdorff in
association with myeloma1 but also associated with other
B-cell neoplasms, including monoclonal gammopathies of undetermined
significance.2-5 By contrast, cryocrystalglobulinemia has
never been reported in mice, although a number of murine monoclonal
cryoglobulins, all of the IgG3 isotype, have been thoroughly
studied.6-10 The exclusive presence of IgG3 within
monoclonal murine cryoglobulins and, also, the enrichment of the IgG3
subclass within mixed cryoglobulins have been ascribed to a strong
tendency of this isotype to self-aggregate due to a more positively
charged CH2 domain.6,10 Monoclonal IgG3 with a wide range
of antigen specificities has been found to cryoprecipitate. Although
the precise mechanisms of cryoprecipitation are not understood, convincing data exist supporting an electrostatic model: 3 constant regions would promote electrostatic interactions and self-aggregation of Ig independently of their antigen-binding site. Besides the role of
constant regions, not all monoclonal IgG3s behave as cryoglobulins and
not all cryoglobulins induce kidney lesions; it is thus clear that
variable region sequences also play a role in self-aggregation, precipitation, and nephritogenicity, noticeably by providing additional positively charged residues. More specifically, residues 6 and 23 of
the heavy (H) chain variable domain have been postulated to play a role
in precipitation in a study comparing 6 IgG3 cryoglobulins with several
noncryoprecipitating monoclonal IgG3s.10 Glutamine and
lysine were found at positions 6 and 23, respectively, in cryoglobulins
and were more positively charged than their counterparts in
noncryoglobulins.10
To study the molecular basis of cryocrystallization, we used a singular
hybridoma clone, 8A4, producing a monoclonal IgG3 antibody against a
hydrogenase from Wolinella succinogenes and spontaneously
forming intracellular crystals. Complete sequences of Ig chains were
determined at the complementary DNA (cDNA) level, looking for unusual V
region sequences that may have been responsible for impaired
trafficking and crystallization within plasma cells and in tissue
deposits. We assayed the in vivo nephrotoxicity of the 8A4 IgG3 and
developed an experimental model of cryocrystalglobulinemia by grafting
8A4 hybridoma cells to mice. Pathologic alterations of mice kidneys
were thoroughly examined. This model also allowed us to show that
limited sequence variation of the V region can convert
an Ig responsible for organized glomerular deposits typical of
cryocrystalglobulinemia with glomerulonephritis into a more classical
serum cryoglobulin, leading to isolated thrombi in vascular lumens like
those seen in the hyperviscosity syndrome, without an inflammatory reaction.
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Material and methods |
Production of hybridomas
Female Balb/c mice (Harlan, Borchen, Germany) were
immunized by intraperitoneal injection of 100 µg of purified
membrane-bound hydrogenase from Wolinella succinogenes (kindly
provided by Dr Achim Kröger, Department of Microbiology,
University of Frankfurt) mixed with the same volume
(100 µL) of adjuvant (ABM-2; Pan-Systems, Aidenbach, Germany). Blood
sera showed positive signals specific against the antigen in an
enzyme-linked immunosorbent assay (ELISA). Boosting was performed 4 times at 4-week intervals using 50 µg of antigen each time.
After the final boosting, the mice were killed and the spleen was
removed aseptically. The spleen cells were fused with P3/NSI/1-Ag4-1 (DSM ACC 145) myeloma cells by using 50% polyethylene glycol 1500 (Roche Diagnostics, Mannheim, Germany). Hybridoma cell selection was
performed with hypoxanthine and azaserine. The cells were cultured and
cloned according to standard protocols.11,12
Standard ELISA
For the standard ELISA, 96-well polystyrene plates (Greiner,
Frickenhausen, Germany) were coated with hydrogenase (1.5 µg/mL) or
antimouse Ig Hybridoma Screening Reagent (Roche Diagnostics) diluted
1:500 in 0.2 mol/L Na2CO3, pH 9.5, for 1 hour
at room temperature. Washing steps were performed using 0.9% NaCl and 0.1% Tween 20. Blocking was done with 1% bovine serum albumin (BSA)
in 50-mM Tris-HCl, pH 7.5; 500 mM NaCl; and 0.1% Tween 20. A total of
50 µL of blood serum, hybridoma supernatant, or purified monoclonal
antibody diluted in 50-mM Tris-HCl, pH 7.5; 500 mM NaCl; and 0.1% BSA
were added to each well and incubated for 1 hour. Bound antibody was
detected with a secondary antimouse IgG antibody (whole molecule)
conjugated to alkaline phosphatase (Sigma, Deisenhofen, Germany),
diluted 1:1000 in blocking buffer. The color reaction was developed
with p-nitrophenylphosphate (1 mg/mL) in 0.5 mM MgCl2, 10%
(vol/vol) diethanolamine, pH 9.8. After 30 or 60 minutes of incubation
at room temperature, signal intensity was determined with an ELISA
reader at 405 nm, using 450 nm as the reference wavelength.
Immunochemical studies
The classes, subclasses, and light (L) chain type of the secreted
antibodies were determined using an immunologic subtyping kit (Roche
Diagnostics) according to the manufacturer's instructions.
Selected clones were grown in standardized conditions at
1 × 106 cells/mL for 24 hours, and Ig production
was measured by ELISA (mouse IgG ELISA; Roche Diagnostics) following
the manufacturer's instructions.
Purification of antibodies was done by applying 50 mL of hybridoma
supernatants to pre-equilibrated protein G affinity columns. Eluted
antibodies were concentrated by ultrafiltration (Centricon, 50 kd
cutoff; Amicon, Beverly, MA) and stored at 4°C.
Purification of crystals from cultured cells was done by lysing cells
in 1% NP40, 0.5% sodium dodecyl sulfate (SDS), 5 mM ethylenediaminetetraacetic acid, 150-mM NaCl, and 50-mM Tris-HCl, pH
8.0, and submitting cell debris to proteinase K (0.75 U/mL for 15 minutes). Centrifugation yielded a pellet only consisting in crystals,
which were washed and resuspended in 0.06 mol/L Tris-HCl, pH 6.8, and
2% SDS.
SDS-polyacrylamide gel electrophoresis (PAGE) was done according to
standard procedures.13 Samples were denatured in sample buffer for 3 minutes at 95°C and subsequently analyzed on a 14% gel. Proteins were transferred onto a polyvinylidine difluoride membrane (Millipore, Bedford, MA). Detection of Ig H and L chains was
done with alkaline phosphatase-conjugated antimouse IgG (whole molecule; Sigma), diluted 1:1000 in blocking buffer, for 1 hour at room temperature.
Purification of the 8A4-NP1 cryoglobulin was done by incubating sera
from 8A4-NP1-grafted mice at 4°C for 1 week; the precipitate was
then washed in cold phosphate-buffered saline (PBS) and redissolved at
37°C in PBS. Agar zone electrophoresis of sera obtained at sacrifice was performed using a Hydrasis apparatus (Sebia,
Issy-les-Moulineaux, France).
Immunofluorescence and microscopy studies on cultured cells
For immunofluorescent intracellular staining, cultured cells were
fixed and permeabilized for 2 minutes in 50% acetone, 50% ethanol and
were air-dried. Staining was carried out with antimouse IgG antiserum
conjugated to fluorescein isothiocyanate (FITC; Sigma). Cells were
examined at a ×400 magnification by epifluorescence.
For confocal fluorescence microscopy, hybridoma cells were plated onto
collagen I-coated glass coverslips. Fixation was performed in 3.7%
(wt/vol) paraformaldehyde. For blocking and permeabilization of the
cells, the coverslips were treated in PBS containing 0.2% (wt/vol) BSA
and 0.2% (vol/vol) Triton X-100. The fluorochrome-labeled secondary
antibody was diluted in blocking buffer at a concentration of 3 µg/mL
for Cy3-labeled antimouse IgG (whole molecule; Sigma, L'Isle d'Abeau,
France) or 15 µg/mL for FITC-labeled antimouse chain and
antimouse chain antibodies (Sigma). Confocal microscopy on a
Sarastro 2000 microscope (Molecular Dynamics, Uppsala,
Sweden) was carried out as described.14
For electron microscopy and immunogold labeling, hybridoma cells were
fixed with 4% paraformaldehyde plus 1% glutaraldehyde. Monoclonal Ig
produced by the cells was labeled with a gold particle-coupled antibody goat antimouse (Amersham, Braunschweig, Germany; 1:5 diluted
with PBS). After washing off unbound antibody (PBS, 4× for
5 minutes each) the material was refixed with 1%
glutaraldehyde. To enlarge the gold particles, the silver amplification
method was applied. Thin sections of resin-embedded cells were cut with the Ultracut S (Reichert, Vienna, Austria),
double-contrasted with uranyl acetate and lead citrate, and viewed in a
Philips 208S electron microscope.
Immunomorphologic analysis in BALB/c mice
Various organ and tumor samples were collected from mice carrying
tumors. Light microscopic examination was performed on multiple slides
stained either with hematoxilin/eosine, Schiff periodic acid, light
green trichrome, silver methenamine, or toluidine blue. Frozen blocks
of all major organs (liver, kidney, spleen, heart) and of tumor
obtained at sacrifice were cut in 4-µm-thick slices. Organs were
studied for the presence of deposited mouse Ig by immunofluorescence
with fluorescein-conjugated polyclonal rabbit antisera against mouse Ig
(Zymed, San Francisco, CA). For ultrastructural studies, small samples
of kidney were fixed in 2.5% glutaraldehyde. Sections were then
stained with uranyl acetate and examined using a Jeol
100 CX electron microscope.
Nucleic acids studies, RACE, sequence analysis, and computing of
molecular models
Total RNA was prepared from the crystal-forming 8A4 clone and its
variants using the RNeasy Kit (Qiagen, Hilden, Germany). Two micrograms
of total RNA were used as templates for the synthesis of
single-stranded cDNA by reverse transcriptase (Superscript II RT, Gibco BRL, Gaithersburg, MD) using an
oligo-dT15 primer (Roche Diagnostics) or a
3-subclass-specific primer. The cDNA was used as a template for
amplification of H and L chain sequences by polymerase chain reaction
(PCR)15 using Thermus aquaticus (Taq) polymerase
(Promega, Madison, WI). The native N-terminal and C-terminal ends of
the cDNA were amplified by 5'- and 3'-RACE (rapid
amplification of cDNA ends) PCR using the 5'/3' RACE kit (Roche Diagnostics). NP91 and NP92 H chain variable region cDNA were
amplified using primers specific for the 5' untranslated region
of the VH558 germline gene segment and for the 1 constant region
gene segment. The V region cDNA was amplified using primers matching the 5' untranslated region of the
V21G and the C gene segment. PCR products
were cloned into pBluescript II KS+ (Stratagene, La Jolla, CA) and
sequenced by the dideoxynucleotide method.16 At least 3 independently obtained PCR products were analyzed by sequencing both
strands. Searches in the EMBL European Molecular Biology Laboratory
database were performed using the FASTA
program.17 The assignment of V(D)J gene
segments was done with the help of the DNAPLOT program at the IMGT
(ImMunoGeneTics) server (http://www.genetik.uni-koeln.de/dnaplot/).
Amino acid-based search, alignment with the Kabat numbering scheme,
and canonic class assignment in the Kabat database18 were
done using the KabatMan software (version 2.19;
http://www.biochem.ucl.ac.uk/~martin/abs/).19 Molecular
modeling of the 8A4 chain and its variant from the 8A4-NP1 clone by
comparison to known Ig sequences was carried out using ProMod
software.20
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Results |
Immunoglobulin production in hybridoma cells
We isolated and studied the 8A4 hybridoma clone producing an IgG3
antibody against the membrane-bound hydrogenase from Wolinella succinogenes. A low secretion rate of mouse IgG3 was estimated by
ELISA on cell culture supernatants of 8A4 (2.3 ± 0.06
µg/106 cells per 24 hours). By contrast, light microscopy
showed that cells cultured at 37°C readily developed abundant and
large rod-shape crystals, which accumulated in living cells (from 1 to
10 crystals per cell) and progressively extended in length,
occasionally protruding beyond the cell surface; crystals also
accumulated in the liquid culture media (Figure
1A). By immunofluorescence or confocal
microscopy, these crystals appeared to include the secreted antibody
and strongly stained with antimouse IgG antisera (Figure 1B, 1C, 1D).
The crystals could also be specifically labeled by immuno-electron
microscopy with a gold particle- (10 nm) coupled antibody goat
antimouse IgG (not shown). Crystals purified from cultured 8A4 cells
were analyzed by SDS-PAGE; Western blotting revealed that they included the monoclonal Ig H and L chain (not shown), and Coomassie blue staining revealed that the monoclonal Ig was the sole component of
crystals (Figure 2A).
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| Fig 1.
In vitro crystal formation in 8A4 cells.
(A) Light microscopy of living cells (original magnification
×400). (B) Immunofluorescence on acetone/ethanol-fixed cells
stained with fluorescein-labeled anti-IgG (original magnification
×400). (C and D) Confocal fluorescence microscopy of 8A4 cells:
hybridoma cell stained with antimouse IgG-Cy3 conjugate. Rod-like
intracellular crystals display strong signals. Intensities are coded by
pseudocolors (C) or brightness (D). Scale bar: 2 µm.
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| Fig 2.
Immunochemical analysis of cell supernatants and
crystals.
(A) SDS-PAGE analysis of crystals formed in vitro by 8A4 cells and
analyzed under nonreducing (lane 1) or reducing (lane 2) conditions;
only polypeptides corresponding to immunoglobulin H and L chains were
detected by Coomassie blue staining. (B) SDS-PAGE analysis under
reducing conditions of antibodies purified from 8A4 (lane 1) and
8A4-NP1 (lane 2); Coomassie blue staining; molecular weight markers are
indicated in kilodaltons. (C) Agar zone electrophoresis of serum from
an 8A4 mouse (lane 1), an 8A4-NP1 mouse (lane 2), and the solubilized
cryoglobulin precipitate obtained from 8A4-NP1 serum (lane 3); the
upper arrow indicates serum albumin, and the bottom arrow marks the
monoclonal cryoglobulin peak.
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The mutated IgG3 8A4-NP1 was obtained as a spontaneous variant of 8A4
that lost production of intracellular crystals but kept secreting an IgG3 antibody that bound hydrogenase. In this
particular variant, Ig secretion raised 7 times to 15.9 ± 1.3
µg/106 cells per 24 hours. When
antibodies secreted by 8A4 and 8A4-NP1 cells were purified and analyzed
by SDS-PAGE, they appeared to include normal-size Ig chains with
similar migrations for the H chains and a slightly faster migration for
the 8A4-NP1 L chain (Figure 2B). Two further subclones without crystal
production (NP91 and NP92) had preserved identical H and L chain
variable domains but had performed a class switch, now both secreting
IgG1.
In vivo pathogenic properties of 8A4 and 8A4-NP1 in mice
Monoclonal IgG3 was produced in vivo in mice injected with the 8A4
or 8A4-NP1 hybridoma (Table 1). Although no
detectable monoclonal peak was observed in 8A4 mice, the 8A4-NP1 IgG3
manifested as an abundant monoclonal peak in sera of 8A4-NP1 mice,
representing more than 50% of total serum protein; this monoclonal
component readily precipitated when sera were incubated in the cold and thus behaves as a type I monoclonal cryoglobulin (Figure 2C). There was
no clinical evidence of cutaneous lesions or arthritis in mice.
In 8A4 mice, light microscopic examination of tumor biopsies showed
abundant crystal inclusions similar to Ig crystals observed in cultured
cells (Figure 3). Contrasting with the
absence of a monoclonal peak, kidney samples showed marked glomerular
lesions with abundant wire-loop deposits along the glomerular basement membrane, predominating in mesangium on the endothelial aspect of the
basement membrane and often resulting in pseudo-thrombi (Figure
4A and 4B); to a lesser extent, deposits
were also found on the epithelial side and occasionally invaded the
urinary space. In some glomeruli, Ig deposits were associated with
endocapillary proliferation and polymorphonuclear leukocyte
infiltration. By immunofluorescence, deposits strongly stained with an
antimouse IgG and were present within all glomeruli (Figure 4C). No
deposits were found in other locations of the kidney or in liver or
spleen. Electron microscopy also confirmed the crystalline nature of
extracellular renal deposits by demonstrating their regular striation
of 12- to 14-nm periodicity (Figure 4D and 4E). The crystals observed in vitro in cultured cells (Figure 1) and in vivo in tumoral cells (Figure 3), either by immunofluorescence staining or by electron microscopy, were identical to those formed in animals as kidney deposits (Figure 4).
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| Fig 3.
Tumors induced by 8A4 cells grafted to mice.
Immunofluorescence microscopy with antimouse Ig conjugate (A, original
magnification ×1000) and light microscopy (B, semi-thin section,
toluidine blue staining, original magnification ×1000) showed
numerous polymorphic crystals located in tumor cell cytoplasms. (C and
D) Electron microscopy showed osmiophilic and highly organized
crystalline inclusions in rough ergastoplasm cisternae with a
periodicity of 12 nm (C, original magnification ×6000; D,
×25 000).
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| Fig 4.
Glomerular lesions in 8A4 mice kidneys.
(A and B) Light microscopy showing the proliferative and exudative
changes (A, original magnification ×200, light green trichromic
staining) and the voluminous depositions of material predominantly in
mesangial and subendothelial areas (B, original magnification
×1000, semi-thin section, toluidine blue staining). (C)
Immunofluorescence microscopy with antimouse Ig conjugate (original
magnification ×200) showed intense staining of deposits strictly
limited to glomeruli without any staining of peritubular capillary
lumen. (D and E) Electron microscopy showed osmiophilic deposits in
mesangial and subendothelial spaces (wire-loop lesion) with few
subendothelial deposits. The glomerular capillary lumens were free of
deposits (D, original magnification ×5000). Note the highly
organized crystalline glomerular deposits formed by densely packed
microtubules (external diameter = 12 nm) in transversal section and
parallel arrays in longitudinal section with a 12-nm striation
periodicity (E, original magnification ×60 000).
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In mice grafted with the 8A4-NP1 hybridoma cells producing
noncrystallizing IgG3, tumor cells did not contain any intracytoplasmic crystalline inclusion (data not shown); neither could crystalline organized deposits be found in glomeruli. Only abundant amorphous Ig
precipitates predominating in glomerular capillary lumens but also in
peritubular capillaries and arteriolar lumens were found without
hypercellularity or polymorphonuclear leukocyte afflux (Figure
5A and 5B). Thrombi (intracapillary
precipitates) were amorphous, without any crystalloid, fibrillar, or
microtubular organization (Figure 5C, 5D, 5E). Numerous cytoplasmic
dense bodies without any ultrastructural organization were found in
glomerular endothelial and epithelial cells and in proximal tubular
cells (Figure 5E).
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| Fig 5.
8A4-NP1 cryoglobulin, renal lesions observed in mice
kidneys.
(A and B) Light microscopy showing intravascular precipitates not
solely in glomerular capillary lumens but also in peritubular capillary
and arteriolar lumens. Note the absence of inflammatory reaction.
Semi-thin sections, toluidine blue staining (A, original magnification
×400; B, ×1000). (C) Immunofluorescence microscopy with
antimouse Ig conjugate (original magnification ×200) showed
staining of intravascular cryoglobulin aggregates. (D and E) Electron
microscopy showed osmiophilic amorphous precipitates in capillary
lumens without crystalline substructure and without any deposits in
subendothelials spaces. Note also the amorphous dense bodies in
epithelial and endothelial cell cytoplasms (D, original magnification
×2500; E, ×12 000).
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Immunoglobulin cDNA sequencing
Sequence of the 8A4 Ig chain cDNAs showed that the VHDJH and
VJ regions had an overall normal
structure (Figure 6). The VH region was
most closely related (87% identical) to the VH558 germline
gene21 rearranged to the DQ52 and JH1 segments, while the
V region was related (95% identical) to
V21G22 and rearranged to J2.
Several unusual substitutions were found, including Gly(+3), Tyr(+7),
Thr(+40), and Met(+106) in the VJ region
and His(+3), Ile(+62), and Val(+85) in the VH region. The VH sequence
also included Gln(+6) and Lys(+23), previously reported as canonic in
murine monoclonal cryoglobulins.10 Ig mRNA sequencing
revealed that 8A4-NP1 only differed from the original 8A4 by a 3-codon
deletion in the V region CDR1 (Figure 6).
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| Fig 6.
Variable region sequences.
Alignment of the primary sequences of 8A4 and 8A4-NP1 proteins with the
germinally encoded VH558/DQ52/JH1 and
V21G/J2 sequences. Dashes indicate
identities. Residues potentially involved in cryoprecipitation or
crystal formation are in bold (unusual residues typical of 8A4) or
underlined (residues shared with other cryoglobulins). Stars indicate
deleted residues. Amino acids are numbered according to
Kabat.18
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Molecular models of the 8A4 and 8A4-NP1 V domains showed
that residues exposed to the solvent differed around the 3-amino acid
deletion, so that Ser(+26) and Glu(+27) preceding the deletion and
Tyr(+28), Gly(+29), and Thr(+30) following the deletion had lateral
chains exposed to the solvent in 8A4 and not in 8A4-NP1. The cDNA
sequences of the constant regions of L and H chain showed no deviation
from the published sequences.23,24
Variable region sequences were also determined from both 8A4 variants,
which had undergone switching to the IgG1 isotype and had
simultaneously lost intracellular crystal formation: For both variants,
VH and V sequences fell in complete identity with those
of 8A4.
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Discussion |
Crystal formation by monoclonal Ig has been reported in various
conditions featuring kidney alterations. Monoclonal L chain crystallization typical of human myeloma-associated Fanconi syndrome has been thoroughly documented at the molecular level: It most often
involves free L chain, and all 4 cases studied at the molecular
level were related to the same VI subgroup gene, O2-O12.25-27 By contrast, molecular
analysis has never been carried out in cases featuring spontaneous
crystallization of complete Ig, and there was no experimental model of
this condition. In human beings, organized aggregates made up of
complete IgG are observed in 2 conditions: myeloma-associated
cryocrystalglobulinemias and glomerulopathies with organized
microtubular monoclonal immunoglobulin deposits (GOMMID), a
complication of chronic lymphocytic leukemia, with monoclonal IgG
forming crystal-like microtubular inclusions in both the leukemic cells
and the kidneys.28 GOMMID can also be observed as a
complication of monoclonal gammopathies without detectable serum cryoglobulin.
To set up a model of murine cryocrystalglobulinemia, we took advantage
of a hybridoma clone spontaneously forming intracellular IgG3 crystals
when cultured at 37°C. When hybridoma cells were grafted into mice,
they yielded tumors that kept producing intracellular crystals in vivo.
Similarly to the situation in human cryocrystalglobulinemia, these mice
readily developed typical crystalline kidney deposits along the inner
aspect of the glomerular basement membrane. Crystalline organized
deposits also led to a strong inflammatory reaction featuring
endocapillary cell proliferation and afflux of neutrophils. Although
considerable amounts of the monoclonal Ig were secreted by the tumor
and apparently circulated through the kidneys to give abundant
deposits, no monoclonal peak was detectable in serum by
electrophoresis, thus indicating a rapid deposition process and
clearance of the protein in its soluble form, even at 37°C in body
fluids. In human cryoglobulinemias also, cryoglobulins that precipitate
at 37°C may be undetectable in serum, and it is well-known that
there is no correlation between the amount of detectable monoclonal Ig
in serum and their ability to yield renal deposits.28,29
By contrast, when mice were injected with tumors secreting the 8A4-NP1
variant, which has lost the production of intracellular crystals, a
strong signal for a monoclonal peak migrating among gammaglobulins
appeared by serum electrophoresis in freshly collected sera from
tumoral mice. This monoclonal Ig disappeared when the serum was kept
for several days at room temperature and behaved as a type I
cryoglobulin, giving an abundant precipitate exclusively made up of
the monoclonal Ig. In those mice, no crystalline deposits were observed in the kidney either; rather, amorphous Ig precipitates developed in vascular lumens of kidneys.
The 8A4 cryocrystalglobulin sequence was strongly related to that of
previously reported murine cryoglobulins, because it belonged to the
IgG3 class and also carried neutral and positively charged glutamine
and lysine residues at positions 6 and 23, respectively.10 Because the 8A4-NP1 Ig only differed from 8A4 by a 3-codon deletion in
the chain CDR1, it also carried the canonic H chain structures previously reported for murine monoclonal cryoglobulins, with a 3
constant region and glutamine 6 and lysine 23.10 Other unusual features shared by 8A4, 8A4-NP1, and all 6 previously sequenced
murine cryoglobulins were the presence of glutamate at position 10, valine at position 18, and alanine at position 71. In fact, the short
CDR1 deletion in 8A4-NP1 not only suppressed crystal formation but
also strongly enhanced in vitro and in vivo Ig secretion (resulting in
a high amount of the monoclonal IgG3 in mice sera) and modified
nephritogenicity of the monoclonal Ig, which only yielded unorganized
intravascular thrombi with no inflammatory reaction. According to
modeling data, the main difference between both monoclonal Ig might be
the loss of some charged amino acids at the surface of the
V domain in 8A4-NP1, which might impede electrostatic
interactions involved both in crystal formation and in affinity of Ig
precipitates for the subentholial space of glomeruli.
Some previously reported murine cryoglobulins were encoded by germline
V genes, and it was suggested that certain pathogenic autoantibodies may be germinally encoded.30 By contrast,
the 8A4 IgG3 carries several mutations and binds a defined
bacterial antigen. It is also clear that the pathogenicity of 8A4
relies on the global structure of the complete Ig rather than
on its antigen specificity. Indeed, crystal formation is lost in
switch variants carrying the very same V regions linked to a 1 H
chain constant region. Strikingly in the 8A4-NP1 variant clone,
mutation of the V region resulted in loss of
crystal formation in the plasma cells without suppressing the antigen
specificity for Wollinella hydrogenase.
Various patterns of glomerular lesions have previously been reported to
be induced by murine cryoglobulins and to be independent from their
antigen binding activity.7,9,30,31 In the case of a
cryoglobulin rheumatoid factor, replacement of the LC with a  1
LC resulted in preservation of cryoglobulin activity and of
glomerulonephritis despite a loss of antigen specificity.30 Neutrophils may play a special role in the development of renal lesions: Certain Ig deposits are able to provoke glomerular neutrophil infiltration and further induce wire-loop glomerular lesions; others do
not promote cell infiltration and solely induce intracapillary amorphous thrombi reminiscent of those seen with
8A4-NP1.30,31
Finally, we have established a murine model of glomerulonephritis
induced by highly organized crystalline monoclonal IgG3 deposits.
Crystal formation in tumoral B cells was correlated with crystalline
organization of IgG3 glomerular wire-loop deposits. Either a
mutation in the L chain or a change in the H chain isotype suppressed
crystal formation within monoclonal B cells. In animals with tumors,
mutation of the V domain resulted in a striking difference in nephritogenicity, with not solely a loss of crystalline glomerular deposits, replaced with amorphous Ig precipitates in vascular lumens, but also the absence of inflammatory reaction.
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Acknowledgments |
We thank Prof Joachim Kirsch for kindly having placed the confocal
fluorescence microscope at our disposal, Dr Roland Pfeiffer for
help with the sample preparation and microscopy, Prof
Philippe Babin for providing us with access to the electron microscope in Poitiers, and Emmanuelle Magnoux, Béatrice Fernandez,
Nathalie Quellard, Josette Nicaud, and Isabelle Poulidor for expert
technical assistance.
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Footnotes |
Submitted November 17, 1999; accepted January 24, 2000.
Supported by grants from Ligue contre le Cancer (Comité
Régional de la Haute-Vienne), Conseil Régional du Limousin,
Association pour la Recherche sur le Cancer (grant 9121), AREN
Poitou-Charentes, PHRC AOM96 058, and INSERM #4R001B. J.-U.R.
supported by grants from the Deutsche Forschungsgemeinschaft (SFB 472),
the Max-Planck-Gesellschaft, and the Fonds der Chemischen Industrie.
S.D. was a recipient from a Société Française de
Néphrologie fellowship.
Reprints: Michel Cogné, Laboratoire
d'Immunologie, CNRS EP118, Centre Hospitalier Universitaire de
Limoges, Institut Universitaire de France, France.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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References |
1.
Von Bonsdorff B, Groth H, Packalen T.
On the presence of a high molecular crystallizable protein in the blood serum in myeloma.
Folia Hematol.
1938;59:184.
2.
Grossman J, Abraham GN, Leddy JP, Condemi JL.
Crystalglobulinemia.
Ann Intern Med.
1972;77:395.
3.
Ball NJ, Wickert W, Marx LH, Thaell JF.
Crystalglobulinemia syndrome: a manifestation of multiple myeloma.
Cancer.
1993;71:1231[Medline]
[Order article via Infotrieve].
4.
Albert L, Inman R, Gordon DA, Chiu B, Amdemichael E, Katz A.
Cryocrystalglobulinemia mimicking rheumatoid arthritis and vasculitis.
J Rheumatol.
1996;23:7.
5.
Papo T, Musset L, Bardin T, et al.
Cryocrystalglobulinemia as a cause of systemic vasculopathy and widespread erosive arthropathy.
Arthritis Rheum.
1996;39:335[Medline]
[Order article via Infotrieve].
6.
Abdelmoula M, Spertini F, Shibata T, et al.
IgG3 is the major source of cryoglobulins in mice.
J Immunol.
1989;143:526[Abstract].
7.
Lemoine R, Berney T, Shibata T, et al.
Induction of "wire-loop" lesions by murine monoclonal IgG3 cryoglobulins.
Kidney Int.
1992;41:65[Medline]
[Order article via Infotrieve].
8.
Itoh J, Nose M, Takahashi S, et al.
Induction of different types of glomerulonephritis by monoclonal antibodies derived from an MRL/lpr lupus mouse.
Am J Pathol.
1993;143:1436[Abstract].
9.
Berney T, Shibata T, Izui S.
Murine cryoglobulinemia: pathogenic and protective IgG3 self-associating antibodies.
J Immunol.
1991;147:3331[Abstract].
10.
Panka DJ, Salant DJ, Jacobson BA, Minto AW, Marshak-Rothstein A.
The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition.
Eur J Immunol.
1995;25:279[Medline]
[Order article via Infotrieve].
11.
Köhler G, Milstein C.
Continuous cultures of fused cells secreting antibody of predefined specifity.
Nature.
1975;256:495[Medline]
[Order article via Infotrieve].
12.
Peters JH, Baumgarten H.
Monoklonale Antikörper: Herstellung und Charakterisierung. 2nd ed. Berlin, Germany: Springer Verlag; 1990.
13.
Laemmli UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature.
1970;227:680[Medline]
[Order article via Infotrieve].
14.
Kirsch J, Betz H.
The postsynaptic localization of the glycine receptor-associated protein gephyrin is regulated by the cytoskeleton.
J Neurosci.
1995;15:4148[Abstract].
15.
Saïki RK, Scharf S, Fallona F.
Enzymatic amplification of  -globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.
Science.
1985;230:1350[Abstract/Free Full Text].
16.
Sanger F, Nicklen S, Coulson AR.
DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci U S A.
1987;74:5463.
17.
Pearson WR, Lipman DJ.
Improved tools for biological sequence comparison.
Proc Natl Sci Acad U S A.
1988;85:2444[Abstract/Free Full Text].
18.
Johnson G, Kabat EA, Wu TT.
Kabat database of proteins of immunological interest. In:
Herzenberg LA,Weir WM,Blackwell C, eds.
Weir's Handbook of Experimental Immunology. 5th ed. Cambridge, MA: Blackwell Science Inc; 1996:6.1-6.21.
19.
Martin ACR.
Accessing the Kabat antibody sequence database by computer.
Proteins.
1996;25:130[Medline]
[Order article via Infotrieve].
20.
Peitsch MC.
ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling.
Biochem Soc Trans.
1996;24:274-279[Medline]
[Order article via Infotrieve].
21.
Yancopoulos GD, Alt FW.
Developmentally controlled and tissue-specific expression of unrearranged VH gene segments.
Cell.
1985;40:271[Medline]
[Order article via Infotrieve].
22.
Alanen A, Weiss S.
Sequence and linkage of the V kappa 21A and G germ-line gene segments in the mouse.
Eur J Immunol.
1989;19:1961[Medline]
[Order article via Infotrieve].
23.
Altenburger W, Neumaier PS, Steinmetz M, Zachau HG.
DNA sequence of the constant gene region of the mouse immunoglobulin kappa chain.
Nucleic Acids Res.
1980;9:971[Abstract/Free Full Text].
24.
Wels JA, Word CJ, Rimm D, et al.
Structural analysis of the murine IgG3 constant region gene.
EMBO J.
1984;3:2041[Medline]
[Order article via Infotrieve].
25.
Aucouturier P, Bauwens M, Khamlichi AA, et al.
Monoclonal Ig L chain and L chain V domain fragment crystallization in myeloma-associated Fanconi's syndrome.
J Immunol.
1993;150:3561[Abstract].
26.
Rocca A, Khamlichi AA, Touchard G, et al.
Light chain V region gene usage restriction and peculiarities in myeloma-associated Fanconi's syndrome.
J Immunol.
1995;155:3245[Abstract].
27.
Decourt C, Rocca A, Bridoux F, et al.
Mutational analysis in murine models for myeloma-associated Fanconi's syndrome or cast myeloma nephropathy.
Blood.
1999;94:3559[Abstract/Free Full Text].
28.
Touchard G, Bauwens M, Goujon JM, Aucouturier P, Patte D, Preud'homme JL.
Glomerulonephritis with organized microtubular monoclonal immunoglobulin deposits. In:
Grünfeld JP,Bach JF,Kreith H,Maxwell MH, eds.
Advances in Nephrology. St. Vol 23. Louis, MO: Mosby Year Book; 1994:149-175.
29.
Touchard G, Preud'homme JL, Aucouturier P, et al.
Nephrotic syndrome associated with chronic lymphocytic leukemia: an immunological and pathological study.
Clin Nephrol.
1989;31:107-116[Medline]
[Order article via Infotrieve].
30.
Reininger L, Berney T, Shibata T, Spertini F, Merina R, Izuis S.
Cryoglobulinemia induced by a murine IgG3 rheumatoid factor: skin vasculitis and glomerulonephritis arise from distinct pathogenic mechanisms.
Proc Natl Acad Sci U S A.
1990;87:10,038-10,042[Abstract/Free Full Text].
31.
Izui S, Fulpius T, Reininger L, Pastore Y, Kobayakawa T.
Role of neutrophils in murine cryoglobulinemia.
Inflamm Res.
1998;47(suppl 3):S145.
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Abstract
Introduction