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Laboratoire d’immunopathologie, Institut d’Hémato-Immunologie, Hôpital Civil, Strasbourg, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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k) producing a natural low affinity
multireactive human autoantibody. These models enable us to test both
the central tolerance mechanisms (reactivity with single-stranded DNA)
and the peripheral tolerance mechanisms after Ag administration. Not
only are the multireactive B cells not deleted in the bone marrow, they
circulate and remain in the periphery even after the prolonged
administration of Ag, the presence of membrane IgD increasing the
number of mature autoreactive B cells. Self-reactive B cells are shown
to be autoantigen ignorant both in vivo and in vitro, but they are not
anergic because they can be easily activated through both B cell
receptor-dependent and -independent pathways. Thus, these mouse lines
reproduce an apparent paradox to the clonal tolerance theory meriting
further investigation of the biological significance of this
phenomenon. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B cell tolerance to soluble Ags has also largely been explored in both tg and non-tg mice. It appears that a number of parameters can affect this complex process and the biology of the self-reactive B cells. For instance, in the case of high affinity BCR for an autoantigen, different outcomes of the B cells have been described depending on the site of Ag encounter. If the Ag is present in the bone marrow, newly generated B cells can be deleted (7), rendered anergic (8, 9, 10, 11), or subjected to receptor editing (12, 13). In contrast, if these high affinity B cells encounter the autoantigen in the periphery, they are deleted (14, 15). Murine models expressing tg BCR with moderate affinity for soluble autoantigens were also studied in the case of rheumatoid factors (RF) (16) and single-stranded DNA (11); the models indicated that anergy is the main tolerance mechanism. However, in addition to the affinity of the BCR and the geography of the Ag encounter that determine the autoreactive B cell fate, the valency of the Ag also appeared critical (17), as well as the cross-reactivity (18).
It is of note that the two last mentioned parameters (extensive cross-reactivity with repetitive determinants on soluble self-Ags) as well as low affinity are the main characteristics of nAAbs. Usually, these Abs are multireactive low affinity IgM encoded by almost unmutated germline variable region genes. They could be the main components of the primary B cell repertoire (19) and their natural escape from the known mechanisms of B cell tolerance in newborns and in adults constitutes an apparent paradox to the clonal tolerance theory. The study of nAAb-producing B cells could focus on related questions relevant both to B cell physiology and to pathologic autoimmunity: 1) how multi self-reactive B cells can escape tolerance check points, and what is their functional state when they reach the periphery; 2) what is the biological function of these nAAb B cells; and 3) are the different autoantigens able (and under what conditions) to drive B cell maturation and lead to the production of potentially pathogenic AAbs?
These questions are not easy to address in a normal polyclonal state. To precisely follow the behavior of multireactive nAAb producing B cells, we generated tg mice that were specifically designed to produce this type of AAb in experimental conditions where both central and peripheral tolerance mechanisms could be tested. The multireactive AAb is a prototype of natural human AAb and reacts with single stranded DNA, thyroglobulin, myoglobin, and human IgG (hIgG; Ref. 20). Here we describe these models and show that nAAb producing B cells are autoantigen ignorant on a nonautoimmune background but can still be activated through BCR-dependent and -independent pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two vectors, pESAC38 and AC38K (provided by K. Rajewsky
and coworkers, University of Cologne, Germany). were modified. These
vectors allow the expression of a murine IgM 
. Restriction sites
SalI and ClaI were introduced in both vectors by
site-directed mutagenesis on both sides of the murine-rearranged
variable region (V) Ig H and L chain genes. The vectors were digested
by SalI and ClaI, and the murine
VH-D-JH and Vk-Jk regions were removed. The human
rearranged V genes originating from a prototype of multireactive nAAb
(Smi) bearing the G6 VH Id and the 17 109 VK Id
(20) were amplified by PCR from the original vectors pRTM1
and PSVG-Vk3 (20) for the light chain. After
amplification, the rearranged H and L chain V genes
(VH-D-JH and Vk-Jk) were subcloned into pESAC38
and AC38K, respectively. The resulting vectors BlSMI (H chain) and
AC38KlSMI (L chain) allow the expression of nAAb Smi variable regions
grafted onto murine constant regions (µ, ). We modified BlSMI to
allow the expression of IgM and IgD. For this purpose, BlSMI was
digested by XhoI and EcoRI, and the resulting
VDJ-Cµ fragment was subcloned into the plasmid pHelix
(Boehringer-Mannheim Biochemicals, Mannheim, Germany). A 12-kb region
XhoI-SmaI originating from the pG13XH vector
(obtained from K. Rajewsky) containing the C
-Cs region was
subcloned into the vector pHelix-VDJ Cµ. The resulting vector was
termed pHelixµSMI. Fig. 1
shows the maps of the different
vectors.
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H and L chain genes coding for the chimeric murine-human nAAb
were excised from vector sequences by EcoRI digestion. The
inserts were purified on a 10–50% sucrose density gradient followed
by extensive dialysis against 5 mM Tris, 0.1 mM EDTA pH 7.5 buffer. The
purified construct was microinjected into pronuclei of fertilized
(C57BL/6 + SJL) F1 zygotes, and these were
transferred to pseudopregnant females to produce the tg mice. Founder
lines were then maintained by backcross mating with the C57BL/6
strain (The Jackson Laboratory, Bar Harbor, ME). H+L tg mice were
obtained by crossing H chain tg mice with the L chain tg mice. Double
tg mice (H+L) were identified by PCR assay of tail DNA
(21). DP10 (µ or µ) tg mice were identified
using the following oligonucleotides: 5'-GGTACAGCAAACTACGCACAG-3'
(SMI H CDR2) and 5'-GCGTCGACTTACCTGAGGAGACGGTGA-3' (SMI HCDR3).
After amplification, these produce a 220-bp fragment. Humkv325 tg mice
were identified using the following nucleotides:
5'-GGGAATTCACCCTGTCTGTGTCTCCAGGGGAAAGAGCC-3' (SMI L FR1) and
5'-TCATCGATTCTACTCACGTTTGATCTCCAC-3' (Vk-Jk junction). These
produce a 260-bp amplification fragment.
Injection of Ags
Mice were i.p. injected with 2 or 4 mg of monomeric hIgG (Novartis, Basel, Switzerland), i.v. injected with 2–8 mg of aggregated (15 min at 65°C) hIgG, or i.v. injected with 8 mg of myoglobin (Novartis).
Quantification of serum IgMa, IgMb, IgMa/17 109, and reactivity with the different Ags
The level of the nAAb in the serum of 4- to 5-wk-old mice was determined by ELISA. Plates were coated with goat anti-mouse IgM (Cappel Laboratories, Durham, NC) at 1 µg/ml, then washed three times. Plates were blocked for 1 h at room temperature with BSA 1%, washed, and incubated with serial dilutions of serum samples 2 h at 37°C. Binding of mouse IgM was determined by adding biotin-labeled anti-mouse IgM (anti-IgMa or anti-IgMb; Cappel Laboratories). After washings, streptavidin-peroxidase (Jackson ImmunoResearch, West Grove, PA) and peroxidase substrate (o-phenylenediamine dihydrochloride; Sigma, St. Louis, MO) were sequentially added. Absorbance was measured at 492 nm. Levels of IgMa or IgMb were determined by comparison with a standard curve using purified IgMa or IgMb. The quantification of IgMa/17 109 double tg Ig was performed by coating the plates with unlabeled anti-mouse IgMa (Cappel Laboratories) and revealing by biotin-labeled monoclonal anti-idiotope 17 109 (0.05 µg/ml). The serum reactivity was tested with human IgG, thyroglobulin, myoglobin, and ssDNA as previously described (20) except that nAAb were revealed with biotin-labeled anti-IgMa.
FACS analysis
Preparation and staining of cells was as follows: spleen cells were isolated by disruption of spleen between glass slides, lysis of RBC with NH4Cl-Tris, then counting. Peritoneal cells were obtained before splenectomy by washing the peritoneal cavity with 3 ml of medium followed by RBC lysis. Cells were stained for 15 min on ice in RPMI 1640 (BioWhittaker, Walkersville, MD) with 2% FCS and 10 mM NaN3. Following washes, when necessary, cells were stained for 15 min with streptavidin-PE (The Jackson Laboratory) at 0.5 mg/ml.
After washes and incubation in propidium iodide, the cells were analyzed using a single laser FACScan flow cytometer (Becton Dickinson, San Jose, CA), and data was collected using the FACS CellQuest program. Only live cells were included in further analysis by excluding propidium iodide-stained cells.
Cell phenotype was determined using the following reagents: FITC
anti-mouse B220 (PharMingen, San Diego, CA), biotin anti-mouse
IgMa or IgMb (PharMingen), biotin anti-mouse IgD (PharMingen),
biotin 17 109 (provided by D. A. Carson, University of San Diego,
San Diego, CA), biotin G6 (provided by Roy Jefferis, University of
Birmingham, Birmingham, England), biotin goat anti-mouse IgM
(Jackson ImmunoResearch), biotin anti-CD19 (PharMingen), biotin
anti-CD23 (PharMingen), biotin AA.4.1 (provided by R. Ceredig,
Basel Institute for Immunology, Basel, Switzerland), biotin goat
anti-mouse light chain (Jackson ImmunoResearch), and
streptavidin-PE (Jackson Immuno-Research).
H IgG binding to tg B cells was tested by incubating C57BL/6 or nAAb tg splenocytes with increasing concentrations of hIgG after CD32 blocking by an anti-CD32 mAb (2.4G2, 1 µg/ml, Fc Block; PharMingen). After washing, cells were labeled with an FITC anti-mouse IgMa (PharMingen) and PE-labeled F(ab')2 of goat anti hIgGFc (Jackson ImmunoResearch).
Immunohistochemistry
Tissues were snap-frozen in optimal cutting temperature medium (Tissue-Tek; Euromedex, Souffelweyersheim, France). Six-micrometer sections were prepared from the tissue blocks and stained with FITC goat anti-mouse IgMa (or IgMb) (PharMingen), FITC goat anti-mouse IgDa (PharMingen), FITC goat anti-mouse CD3 (PharMingen), and biotin 17 109 followed by streptavidin-PE (Jackson ImmunoResearch). Briefly, 100 µl of medium or an optimal dilution of the reagent was added to each slide and incubated for 2 h on ice. Slides were then washed with PBS (pH 7.2) before adding 100 µl of streptavidin-PE. The slides were then washed before mounting.
Generation of nAAb-secreting hybridomas
Spleens were removed from µk tg mice and then teased
apart in serum-free IMDM (Irvine Scientific, Santa Ana, CA). Spleen
cells (108) were combined with nonsecreting
myeloma SP2O cells, washed in serum-free medium, and then pelleted.
Polyethylene glycol 1500 (0.8 ml; Boehringer Mannheim) was added slowly
to the pellet while stirring. After 2 min, the cells were diluted
slowly with medium, washed, and then plated out in four 24-well plates
at 1 ml/well in IMDM plus 10% FCS. After 24 and 48 h, the wells
were fed with medium supplemented with HAT (hypoxanthine, thymidine,
aminopterin; Sigma). Fusion culture supernatants were tested for the
presence of the nAAb between days 14 and 21. Positive wells were then
cloned and positive monoclonal hybridomas were selected by ELISA on the
basis of 17 109 expression. Multireactivity of nAAb-tg hybridomas was
tested as previously described (22, 23), except that
biotin-labeled 17 109 was used to detect the binding of the chimeric Ab
and of original Smi Ab.
B cell proliferation assays
Spleens were removed from tg mice, non-tg littermates, or control C57BL/6 mice and teased apart. Cells were washed and resuspended at 2.106/ml in RPMI 1640 (BioWittaker) with the addition of antibiotics, glutamine, 50 µM 2-ME, and 10% heat-inactivated FCS. Cells (2 x 105) were added to each well of a round-bottom 96-well plate in a total volume of 100 µl. Another 100 µl of medium was then added containing one of the following reagents: LPS from Salmonella typhosa (Sigma) at 50 µg/ml, goat anti-mouse IgM (Jackson ImmunoResearch), soluble or aggregated human IgG (Novartis) at different concentrations (0–20 mg/ml). Aggregated human IgG were prepared by heating hIgG at 65°C for 15 min.
Proliferation was measured on day 3 by adding 200 µl of medium containing 10% Alamar Blue (Interchem, Paramus, NJ). After 4 h, absorbance was measured at 590 nm using the Fluorite 1000 reader (Dynatech Laboratories, Chantilly, VA).
After in vitro stimulation with anti-IgM, LPS, or hIgG, the phenotype of the cells was determined by FACS analysis. Briefly the cells were stained using FITC goat anti-mouse IgMa (PharMingen), biotin goat anti-mouse CD86, or biotin anti-MHC class II (PharMingen), washed, and labeled with streptavidin-PE. The cells were incubated in propidium iodide and analyzed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). nAAb expressing B cells are not deleted
By crossing mice expressing nAAb VH (µ or
µ) and mice expressing nAAb Vk, we obtained nAAb double-tg mice
(µk and µk), which were analyzed at 4–6 wk of age.
Approximately 35% (µk) and 32% (µk) of total splenocytes
express the AAb variable regions. The tg µ-chain expression can be
detected by both anti G6 Id and anti-µa reagent, which were used
indifferently in subsequent experiments. Fig. 2 clearly shows that in µk mice,
both in the bone marrow and in the spleen, the vast majority of
B220+ B cells coexpresses the tg H and L chains,
respectively, µa+ and 17
109+. Similar results were obtained with µk
mice (data not shown). Only 2% of the bone marrow cells and 3% of the
splenocytes express endogenous µb H chains in µk nAAb mice
compared with 3% (bone marrow) and 3% (spleen) in µk nAAb mice,
showing that the allelic exclusion was almost complete. The maturation
of the tg µk B cells from the bone marrow to the spleen is
associated with an increased expression of a and CD23, but a
decreased expression of AA4.1 as expected. Table I describes the phenotype of bone marrow,
and splenic and peritoneal B cells in these mice. Thus, it appears that
nAAb-expressing B cells are not deleted and join the periphery. The
precise localization of the nAAb B cells in primary follicles was
determined by immunohistology (Fig. 3).
They are strictly localized in the B cell zones and were never detected
(µk or µk) within the germinal centers of secondary follicles or
within the T cell zones, which could have indicated Ag-driven
activation and expansion.
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Grafting Smi variable regions onto murine constant regions of the
H and L chains of Ig could modify the reactivity profile of the Ab.
Therefore, we checked: 1) the serum reactivity of the IgMa; 2) the
light chain Id expression of these IgMa; and 3) the ability of the tg B
cells to bind hIgG. Finally, we generated hybridomas from tg splenic B
cells. The serum reactivity of nAAb tg mice is shown in Fig. 4. At a serum concentration of 5 µg/ml
of IgMa, the nAAb tg serum reacts with hIgG and slightly with ssDNA.
Considering the Id expression, the levels of serum tg IgM were very
similar in nAAb tg mice when the ELISA quantifications were performed
using as a second reagent an anti-IgMa or 17 109, implying that the
tg H chain was only associated with the tg light chain (data not
shown). We also checked the ability of nAAb tg B cells to bind hIgG.
After CD32 blocking, and despite the low affinity of the nAAb, a
concentration-dependant discrete binding of hIgG was detectable on
IgMa-bearing B cells in tg splenocytes, as shown in Fig. 5. Finally, we generated hybridomas from
tg splenic B cells; these monoclonal chimeric Ab (both G6 and 17 109
positive, data not shown) reacted with ssDNA, human thyroglobulin,
human myoglobin, and human IgG in a manner similar to the original
human Smi Ab (Fig. 4).
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H chain on nAAb tg B cells seems to favor B
cell maturation
Central tolerance mechanisms operate at the B cell stage where
IgM, but not IgD, is expressed. The comparison of the two tg lines,
µk and µk, showed that µk tg B cells were neither deleted nor
arrested at the pre-B stage. However, in the periphery, the percentage
of tg B cells that expresses an immature phenotype (AA4.1 positive,
CD23 negative) is higher in µk nAAb mice compared with µk mice
despite nearly equivalent absolute numbers of total B cells (Table I).
Although not significantly different, the serum levels of IgMa seem
higher in µk mice compared with µk mice (Table II). To find out whether the phenotype of
tg µk mice was linked to the autoreactivity of the BCR, we
analyzed mice lacking the light chain transgene and, yet again,
observed an increased mature B cell pool in µ vs µ tg mice,
showing that this effect was independent of the specificity of the BCR
(data not shown).
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The lack of deletion of nAAb tg B cells in these mouse lines,
despite the reactivity with ssDNA, could be related either to the weak
affinity of the BCR for this Ag, the Ag valency, or its concentration
in the vicinity of the tg B cells. Smi nAAb being multireactive, it was
possible to test high concentrations of two different Ags on the tg B
cell populations after in vivo Ag injection. Different modes of Ag
administrations (i.p., i.v.), different doses of Ag from 2 to 8 mg (to
the extent of mimicking human serological levels of IgG), different
forms of Ag (monomeric IgG, heat-aggregated IgG), and different Ags
(human IgG and myoglobin) were tested in both µk and µk
lines.
For instance, when heat-aggregated hIgG (8 mg) was i.v. injected in
µk mice (Table III) we did not
observe any modification of the tg B cell populations 2 and 5 days
after Ag administration. In particular, Ag administration, no matter
what autoantigen, does not lead to the deletion of autoreactive B cells
nor to their activation either in the bone marrow or in the spleen. It
does not induce a maturation arrest in the bone marrow, the
fluorescence intensity of the tg mb IgM was not modified (data not
shown), and tg B cells were not activated as judged by their unchanged
expression of CD54, CD86, CD44, and MHC class II molecules (Fig. 5). We
observed the same negative results after myoglobin administration.
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Autoantigen ignorant but not anergic
There is no general consensus on the exact phenotype of anergic B
cells, but a down-regulation of mb IgM was previously reported in the
HEL-anti-HEL tg model (8). The density of mb IgMa and
mb IgDa was equivalent in tg nAAb B cells, in non-tg IgMb-bearing B
cells, in single tg µ and µ B cells, and in control IgMa
allotype BALB/c mice (data not shown). Peripheral administration of
human IgG or myoglobin did not modify the density of mb IgMa on the
surface of tg B cells. The levels of secretion of tg IgMa (13 ± 8
µg/ml in µk mice ; 32 ± 20 µg/ml in µk mice) were not
modified 2 and 5 days after in vivo Ag administration. These results
were identical in µk and µk mice.
In vitro tg B cells were equally Ag ignorant. Considering that
heat-aggregated human IgG were able to cross-react with Fc
RII on tg
B cells and, by cross-linking the BCR, inhibit B cell activation
(24), we also tested various concentrations of monomeric
human IgG and myoglobin. They were also unable to activate the tg B
cells (data not shown). However, when stimulated with bacterial LPS (50
µg/ml) or anti-IgM (10 mg/ml) splenic µk and µk tg B cells
were readily able to proliferate and to be activated showing that
BCR-dependent and -independent pathways of B cell activation were
functional (Figs. 6 and 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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k) the
level of endogenous IgMb. Whether this low secretion of tg Ig nAAb
represents a low degree of undetectable B cell activation or is a
consequence of a " leaky " tg B cell phenotype is unknown. However,
it is of note that we were unable to detect any sign of tg B cell
activation, even after peripheral Ag injections. The role of mb IgD in B lymphocyte development was previously studied in different models. Tg mice expressing mb IgD and IgM have an increased mature B cell pool (29) compared with tg mice expressing only mb IgM. Our results, even though originating from single IgM and IgM/IgD tg lines, are consistent with these data and those of others (30). nAAb B cells exit from the bone marrow whether they express IgD or not, but the proportion of peripheral immature B cells (AA 4.1 positive, CD23 negative) is significantly higher in tg mice that express only IgM. The autoreactive nature of the tg BCR in our mice is probably not responsible for the increased maturation of IgD-positive B cells because the same phenomenon was observed in mice lacking the light chain tg and, consequently, having a diverse B cell repertoire.
Other tg models have tested the history of autoreactive B cells against soluble Ag. Each of these models seems peculiar to itself and each could answer different questions. Tg mice expressing high affinity autoantibodies (AAb) were described and did not always confirm the results originating from the initial HEL-anti HEL model (2). Anti dsDNA B cells and RF B cells were either deleted (7, 31), rendered anergic (10), or subjected to receptor editing (32) during their bone marrow maturation. Our nAAb being a prototype of natural G6+/17 109+ RF, it is interesting to compare our data with the other RF tg models. Monoreactive high affinity human RF that did not encounter the Ag during ontogeny were deleted when they did encounter their Ag (hIgG) after i.p. injection (15). Mouse intermediate affinity RF B cells on a nonautoimmune background show fairly similar behavior to our nAAb models; these B cells are not deleted but their functional state seems to be related to a certain degree of Ag-specific activation as judged by spontaneous RF secretion (16). In our models, we do not think that the nAAb secretion is linked to Ag-specific B cell activation because we also observed IgMa secretion in tg mice lacking the tg light chain. Even large quantities of Ag, delivered in periphery, were unable to activate these B cells and to induce nAAb secretion. Considering our in vitro results, which show that nAAb B cells are not activated after human IgG or myoglobin contact, these models are close to, but somewhat different from, tg anti ssDNA where B cells are functionally inactivated, have a decreased total surface Ig, are distributed throughout the B cell follicle, are phenotypically mature, and are responsive to LPS and anti-IgM stimulations (33, 34).
The mechanism by which nAAb B cells escape tolerance (deletion or anergy) is probably related to the affinity of the BCR. It was previously shown that most B cells that can bind an Ag can be neither stimulated nor tolerized by this Ag, even when the former is highly concentrated (35). Thus, it seems that only B cells bearing BCR with a relatively high affinity for a self-Ag would be eliminated. B cells with a very low affinity for self-Ags will not reach the affinity threshold for tolerance induction. The recent description of the different fates of anti-dsDNA B cells in a single tg line is in accord with the notion of a molecular threshold for tolerance induction (36). This notion also questions the potential roles of these nAAb B cells in general, and of natural RF B cells in particular, in adult mice. These cells are autoantigen ignorant because both in vivo and in vitro they are unable to be activated by human IgG (monomeric and multimeric) or myoglobin. However, they can be activated after anti-IgM treatment in vitro, clearly showing that the BCR-dependent pathway is normal in these B cells. They can also be activated through a non-BCR pathway, after LPS stimulation. We could hypothesize that lymph node nAAb B cells, through their ability to cross-react with foreign Ag on different microorganisms with higher affinities, may be activated to secrete Ab and undergo H chain switch and variable region mutations. This mechanism can be considered a first line of defense against pathogens, but could also be responsible for the generation of high affinity pathogenic AAb in the presence of T cell help.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-Louis Pasquali, Institut d’Hémato-Immunologie, 1 place de l’hôpital, Hôpital Civil, 67091 Strasbourg Cedex, France.
3 Abbreviations used in this paper: nAAb, natural autoantibodies; tg, transgenic; mb, membrane; BCR, B cell receptor; RF, rheumatoid factor.
Received for publication June 23, 2000. Accepted for publication October 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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membrane heavy chains of Ig. Int. Immunol. 6:1905.This article has been cited by other articles:
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