The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steeves, M. A.
Right arrow Articles by Marion, T. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steeves, M. A.
Right arrow Articles by Marion, T. N.
The Journal of Immunology, 2004, 172: 6568-6577.
Copyright © 2004 by The American Association of Immunologists

Tolerance to DNA in (NZB x NZW)F1 Mice That Inherit an Anti-DNA VH as a Conventional µ H Chain Transgene but Not as a VH Knock-in Transgene1

Meredith A. Steeves and Tony N. Marion2

Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lupus-prone (NZB x NZW)F1 (BWF1) mice were made transgenic (Tg) for an anti-DNA Ab inherited either as a conventional VH3H9-µ H chain Tg (3H9-µ) with or without a conventional V{kappa}8-{kappa} Tg, or a VH3H9 VH knock-in Tg allele (3H9R) with or without a V{kappa}4 V{kappa} knock-in Tg allele (V{kappa}4R). VH3H9 yields an anti-DNA Ab with most L chains including an anti-ssDNA with the V{kappa}8 Tg and an anti-dsDNA with the V{kappa}4 Tg. BWF1 mice that inherited the conventional 3H9-µ had normal serum IgM, little to none of which was encoded by 3H9-µ, and only a small percentage of those mice had serum anti-DNA, none of which was transgene encoded. B cells expressing the conventional 3H9-µ Tg were anergic. BWF1 mice that inherited the knock-in 3H9R Tg allele also had normal serum IgM, one-half of which was encoded by 3H9R, and produced anti-DNA encoded by the Tg allele. Most B cells expressing the knock-in 3H9R Tg also had an anergic phenotype. The results indicate that autoimmune-prone BWF1 mice initially develop effective B cell tolerance to DNA through anergy, and anergy was sustained in 3H9-µ Tg peripheral B cells but not in 3H9R Tg B cells. B cells expressing the 3H9R knock-in Tg allele were able to achieve an activation threshold that B cells expressing the 3H9-µ conventional Tg could not. The maintenance of B cell tolerance to DNA in autoimmune-prone BWF1 mice appears to differ from both normal mice and autoimmune-prone MRLlpr/lpr mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE)3 is characterized in part by autoimmunity to DNA (1), as well as other nuclear autoantigens (2). A consistently elevated anti-DNA Ab titer is a major diagnostic criterion in clinical assessment of potential SLE patients. In accordance, IgG anti-DNA Abs have been found deposited in the glomeruli of diseased human kidneys (3, 4) and, when eluted, exhibit specificity for dsDNA. (NZB x NZW)F1 (BWF1) mice are the classic animal model for SLE and closely mimic the human autoimmune disease with respect to autoantibody production, sex distribution, and the pathology of lupus nephritis (5, 6). BWF1 mice spontaneously develop Abs to dsDNA, as well as other nuclear autoantigens, that contribute to severe glomerulonephritis and premature death at 8–10 mo. Autoreactive anti-DNA B cells are present in the periphery of both autoimmune and nonautoimmune mice (7) and can be activated to produce anti-DNA Ab (8); however, in BWF1 autoimmune mice, the spontaneous activation of anti-DNA B cells is sustained (9, 10). The autoimmune response to DNA in BWF1 mice has all of the characteristics of an Ag-driven, clonally selected, secondary immune response (11). The relevant immunogen for the Ag-driven autoimmune response must be DNA, structures that contain DNA, or, alternatively, structures on apoptotic cells or blebs that bind anti-DNA (12). Anti-DNA Abs with the same structural and pathogenetic characteristics as autoimmune anti-DNA can be induced by deliberate immunization (13) or virus inoculation (14) but only transiently. Induced autoimmunity to DNA in nonautoimmune mice is characterized by low autoantibody titers and lack of a true memory B cell response (15). Thus, it is apparent that self-reactive B cells in normal mice are under active, negative regulation. The high titers of anti-DNA, as well as other autospecificities seen in BWF1 mice, indicate that a breakdown in normal immunological tolerance to self has occurred. Several regulatory mechanisms control self-reactive B cells including deletion (16, 17), anergy (18, 19), and receptor editing (20, 21, 22, 23).

To better understand how tolerance to DNA is lost in autoimmune-prone BWF1 mice and more closely dissect the development of the autoimmune anti-DNA response, NZW and NZB mice transgenic (Tg) for different rearranged anti-DNA VH and VL genes, respectively, were generated by backcross breeding. NZW mice inherited either the rearranged VH gene from the 3H9 anti-DNA hybridoma (VH3H9) (24) or the rearranged VH gene from an anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) Ab, H50G (VHNP) (25). VH3H9 was inherited as either a site-directed JH knock-in (3H9R) (26) or a conventional, randomly inserted µ H chain (3H9-µ) (19). VHNP was inherited as a µ H chain conventional Tg (VHNP-µ) (27). NZB mice inherited either the rearranged V{kappa}8-J{kappa}5 of an anti-influenza hemagglutinin Ab as a conventional {kappa} Tg (28) (V{kappa}8-{kappa}) or the rearranged V{kappa}4-J{kappa}4 from 3H9 as a J{kappa} knock-in (22) (V{kappa}4R). Although the 3H9R VH Tg can be class-switched and the 3H9R and V{kappa}4R Tgs can be somatically mutated and replaced by receptor editing, the conventional 3H9-µ and V{kappa}8-{kappa} Tgs cannot be class-switched or edited. VH3H9 with V{kappa}8 produces an anti-DNA with ssDNA specificity (29), VH3H9 with V{kappa}4 produces an anti-DNA with dsDNA specificity, and VHNP-V{kappa}8 yields an Ab with unknown specificity. Previous studies have used BWF1 mice Tg only for the H chain of an anti-DNA Ab (30, 31) or a knock-in of the VH for an anti-DNA (32), and each of the previous studies used a different anti-DNA VH Tg. The present study is the first to compare B cell development and anti-DNA Ab production in autoimmune-prone BWF1 mice Tg for the VH of an anti-DNA Ab inherited either as a JH knock-in or as a conventional H chain Tg.

BWF1 mice Tg for 3H9-µ or 3H9-µ-V{kappa}8-{kappa} produced no anti-DNA even though mice from both Tg groups had normal total serum IgM and IgG and IgG anti-nuclear Ab (ANA). Very little of the serum Ig was derived from the 3H9-µ Tg, and B cells expressing Tg-encoded IgMa appeared to be anergic. Serum Igs and IgG anti-DNA in V{kappa}8-{kappa} and Tg– littermates were similar to those in normal BWF1 mice. The majority of B cells expressing IgMa encoded by the 3H9R knock-in, with or without a V{kappa}4R encoded L chain, had an anergic phenotype; nevertheless, the respective BWF1 mice had serum IgM and IgG anti-DNA similar to that in normal, non-Tg BWF1 mice. The differences in B cell phenotype and Ab production between conventional and knock-in Tg BWF1 mice offer interesting insight into tolerance and autoimmunity to DNA in these autoimmune-prone mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female NZB and male NZW mice were obtained from Harlan (Indianapolis, IN) and housed in a specific pathogen-free facility at the University of Tennessee Health Science Center, Memphis, TN. VH3H9-µ (19) and V{kappa}8-{kappa} Tg (28) BALB/c mice were gifts from Dr. J. Erikson (Wistar Institute, Philadelphia, PA). VH3H9R (26) and V{kappa}4R (22) Tg BALB/c mice were gifts from Dr. M. Weigert (University of Chicago, Chicago, IL). C.B-17 mice Tg for a conventional H chain Tg (VHNP-µ) associated with an anti-NP Ab (27) were generously provided by Dr. G. Kelsoe (Duke University, Durham, NC). Mice Tg for the VHNP Tg which has a VDJ with VH186.2-DFL16.1 with an H50G mutation were generated by Dr. M. Shlomchik (Yale University, New Haven, CT). VH Tgs were backcrossed into NZW mice for a minimum of five (VH3H9R, VHNP) or eight (VH3H9-µ) generations, respectively. VL Tgs were backcrossed into NZB mice for at least five (V{kappa}4R) or eight (V{kappa}8) generations, respectively. All Tgs were obtained on BALB/c or C.B-17 backgrounds. Single crosses of Tg NZB with Tg NZW were performed to obtain BWF1 mice with one of nine possible Tg genotypes: 3H9-µ-V{kappa}8-{kappa}, 3H9-µ, V{kappa}8-{kappa}, or Tg–; 3H9R-V{kappa}4, 3H9R, V{kappa}4R, or Tg–; and VHNP-µ-V{kappa}8-{kappa}, VHNP-µ, V{kappa}8-{kappa}, and Tg– (Table I). All of the H chain Tgs had the Igh-6a allele and were expressed as an IgMa allotype. Endogenous H chain rearrangements were on the Igh-6b allele and were IgMb. IgMa and IgMb were distinguished with appropriate allotype-specific Abs.


View this table:
[in this window]
[in a new window]
  		Table I. VH and VL transgenes inherited by BWF1 mice

 
PCR was used to determine the Tg genotype of parental and hybrid F1 mice (19, 26, 27, 33) Tail clips of 1–2 mm were collected into 50 µl of lysis buffer. Proteinase K (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 0.1 mg/ml; samples were incubated at 56°C overnight and then stored at –20°C. PCR primer pairs were as follows: 5' VH3H9 sense primer 5'-CTGTCGGAACTGCGGTAAGT-3' with 3' antisense VH3H9-CDR3 primer 5'-CATAACATAGGAATATTTACTCCTCGC-3' for VH3H9-µ and VH3H9R, 5' sense 5'-TCTAGAATTCAGGTCCAACTGCAGCAGCC-3' and 3' antisense 5'-ACGGATCCTGTGAGAGTGGTGCCT-3' for VHNP, 5' V{kappa} leader sense primer 5'-GGTACCTGTGGGGACATTGTG-3' and 3' J{kappa}5 antisense primer 5'-AGCACCGAACGTGAGAGG-3' for V{kappa}8-{kappa}, and 5' V{kappa} consensus primer 5'-GACATTCAGCTGACCCAGTCTCC-3' and 3' CDR3 primer 5'-GAACGTGAATGGGTAACCACACC-3' for V{kappa}4R. {beta}2-microglobulin was amplified from all tail clip samples as an internal control for DNA quality with sense 5'-CAAGTATACTCACGCCACCC-3' and antisense 5'-CCACACAGATGGAGCGTCCAG-3' primers. All Tgs except V{kappa}4R were amplified using 95°C for 1 min, 55°C for 2 min, and 72°C for 1 min for 39 cycles. V{kappa}4R was amplified with 95°C for 1 min, 66°C for 30 s, and 72°C for 1 min for 35 cycles.

Serum Ig and anti-DNA assays

Anti-DNA Abs in mouse sera were measured as described previously (34). Briefly, ELISA plates (Immunlon I; Dynex Technologies, Chantilly, VA) were coated with 10 µg/ml purified, sonicated (0.5–3 kb) calf thymus DNA (Sigma-Aldrich) in PBS. Bound serum Abs in titrated sera were detected with biotinylated mAbs to IgMa or IgMb (BD PharMingen, San Jose, CA) or biotinylated goat anti-mouse IgG or IgM (Southern Biotechnology Associates (SBA), Birmingham, AL) and streptavidin-alkaline phosphatase (SBA). Total serum Ig levels were similarly determined using plates coated with 1 µg/ml goat anti-mouse IgM for total IgMa and IgMb or 1 µg/ml goat anti-mouse Ig for total IgM or IgG determination. Serum titers are the reciprocal of the dilution that gave 50% maximal binding for the plate. ANAs in mouse sera were detected with acetone-fixed HEP-2 cells (Inova, San Diego, CA). Briefly, HEP-2 slides were warmed to room temperature for 10 min and then incubated with a 1/3 dilution of mouse serum for 30 min in a wet box. Bound Ab was detected with FITC-conjugated goat anti-mouse IgG (SBA). Pooled normal BALB/c mouse serum was used as a negative control. Data were statistically analyzed by ANOVA with Kruskal-Wallis post test for nonparametric and Student-Newman-Keuls multiple comparisons test for parametric data using GraphPad InStat version 3.05 (GraphPad Software, San Diego, www.graphpad.com).

Flow cytometry

Murine spleen and bone marrow cells were collected, blocked with goat and rat IgG (Sigma-Aldrich), both at a final concentration of 0.2 mg/ml, and incubated with one to three fluorochrome-conjugated Abs. Stained cells were analyzed on a BD FACScan (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences). The following Abs were used: FITC, PE, or biotin-conjugated anti-CD45R/B220 (0.5 µg/106 cells) (SBA); PE or biotin-conjugated anti-IgD (0.05 or 0.5 µg/106 cells) (SBA); FITC-conjugated anti-IgM (0.5 µg/106 cells) (SBA); biotin-conjugated anti-CD24/HSA (1 µg/106 cells; SBA); PE-anti-IgMb (0.125 µg/106 cells; BD PharMingen, San Diego, CA), or FITC-IgMa (1.0 µg/106 cells) (BD PharMingen). Biotin-conjugated Abs were detected with streptavidin-PerCP (BD Biosciences). A minimum of three individual adult mice per genotype were analyzed. Ages were between 6 wk and 9 mo.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Longevity and absence of autoimmune disease in Tg BWF1 mice

B cell development and autoantibody production were analyzed in Tg BWF1 mice that inherited anti-DNA Ab production as a conventional H chain Tg with or without a conventional L chain Tg or a knock-in VH Tg allele with or without a knock-in V{kappa} Tg allele (Table I). As noted above, B cell receptors and Abs encoded by conventional Tgs cannot be edited or isotype switched, but they can be eliminated, presumably as a consequence of attempted editing. B cell receptors and Abs encoded by VH and V{kappa} knock-in Tgs can be edited, somatically mutated, and isotype switched. VH3H9 combines with many different L chains to produce an anti-DNA Ab (35). Mice that inherited both the conventional 3H9-µ H and the V{kappa}8-{kappa} L Tgs were monospecific for an anti-DNA that has relatively low affinity for DNA and binds ssDNA but not dsDNA. Mice that inherited both the knock-in 3H9R VH and the V{kappa}4R V{kappa} were monospecific for an anti-DNA that binds dsDNA with relatively high affinity. The experiments also analyzed B cell development and autoantibody production in BWF1 that inherited a conventional H and/or L chain Tg for an Ab with no known DNA or other autoreactivity.

All BWF1 Tg– littermates developed autoimmune disease and had life spans similar to those of BWF1 mice derived from matings of inbred NZB and NZW mice (data not shown). The Tg– mice produced IgG anti-DNA by 6 mo of age and developed glomerulonephritis with proteinuria and elevated blood urea nitrogen and died by ~10 mo of age. V{kappa}8-{kappa} and V{kappa}4R Tg BWF1 mice had delayed development of autoimmune disease and prolonged life compared with Tg– BWF1 mice. V{kappa}8-{kappa} and V{kappa}4R Tg BWF1 mice lived as long as 18 mo unless terminated for experiments. 3H9-µ-V{kappa}8-{kappa}, 3H9-µ, 3H9R-V{kappa}4R, and 3H9R Tg BWF1 mice had no signs of glomerulonephritis detected as proteinuria or elevated blood urea nitrogen up to 1 year of age at which time most were terminated. 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice up to 15 mo of age still had no signs of autoimmune disease. Although VHNP-µ-V{kappa}8-{kappa} and VHNP-µ Tg BWF1 never had any signs of autoimmune disease, they were highly susceptible to the development of eye infections after retro-orbital puncture for blood collection. The infections were resolved with antibiotics. Susceptibility to infection in VHNP-µ and VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice was likely due in part to the absence of isotype switched Ig, e.g., IgA, in those mice (see below).

Tolerance to DNA in conventional 3H9-µ-V{kappa}8-{kappa} but not site-directed 3H9R-V{kappa}4R Tg mice

Conventional 3H9-µ-V{kappa}8-{kappa} and 3H9-µ Tg BWF1 mice differed remarkably from 3H9R-V{kappa}4R and 3H9R knock-in Tg BWF1 mice with respect to the production of Tg-encoded IgMa anti-DNA. Only 2 of 26 3H9-µ-V{kappa}8-{kappa} and 4 of 16 3H9-µ Tg BWF1 mice developed detectable serum IgM or IgG anti-DNA even after 9–12 mo of age (Fig. 1, B and D). By 14 mo of age, three of five 3H9-µ-V{kappa}8-{kappa} and four of four 3H9-µ Tg BWF1 mice were producing IgG anti-DNA (geometric mean titers (SD), 238 ± 1.73 and 448 ± 3.12, respectively), but even after 14 mo of age, none of the mice produced Tg-encoded, IgMa anti-DNA (not shown). Conventional 3H9-µ-V{kappa}8-{kappa} and Tg 3H9-µ BWF1 mice were autoimmune because most of the sera assayed from 3H9-µ-V{kappa}8-{kappa} and 3H9-µ Tg BWF1 mice had IgG ANA with nucleolar and/or speckled nuclear staining patterns on Hep-2 cells (Fig. 2 and Table II). Knock-in 3H9R-V{kappa}4R and 3H9R Tg BWF1 mice, in contrast, developed autoimmune IgM and IgG anti-DNA and a homogeneous ANA (Fig. 2 and not shown) similarly to non-Tg littermate BWF1 mice (Fig. 3, B and D) (p > 0.05). Most of the IgM anti-DNA in 3H9R-V{kappa}4R and 3H9R BWF1 was Tg-encoded IgMa (p < 0.01, IgMa vs IgMb anti-DNA). Both conventional L chain only V{kappa}8-{kappa} (Fig. 1, B and D) and knock-in L chain only V{kappa}4R Tg BWF1 mice (Fig. 3, B and D) had serum IgM and IgG anti-DNA titers similar to those in Tg– littermates (p > 0.05).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 1. Total serum Ig and anti-DNA in 3H9-µ-V{kappa}8-{kappa} and 3H9-µ, V{kappa}8-{kappa}, and Tg– littermate BWF1 mice. A, Total serum IgM; B, serum IgM anti-DNA; C, total serum IgG; D, serum IgG anti-DNA. Titers for total IgM and IgG and for IgM and IgG anti-DNA are indicated. Titers were measured as reciprocal serum dilutions that yielded 50% maximum binding by ELISA. Each point represents an individual mouse. Ages were ~6 mo. x, IgMa levels in pooled normal BALB/c sera; +, IgMb levels in pooled unimmunized NZW sera.

 


View larger version (115K):
[in this window]
[in a new window]
  		FIGURE 2. ANA immunofluorescence on Hep2 cells. A, Tg– BWF1 mice; B, BALB/c mice; C and D, 3H9-µ-V{kappa}8-{kappa} BWF1 mice. Sera were diluted 1/3. 3H9R-V{kappa}4R BWF1 mice produced ANA pattern similar to A.

 

View this table:
[in this window]
[in a new window]
  		Table II. Serum ANA in 3H9-µ and V{kappa}8-{kappa} Tg BWF1 mice

 


View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3. Total serum Ig and anti-DNA in 3H9R-V{kappa}4R and 3H9R, V{kappa}4R, and Tg– littermate BWF1 mice. For details, see legend to Fig. 1.

 
Anergy in IgMa+ B cells in conventional 3H9-µ and knock-in 3H9R but not VHNP-µ conventional Tg BWF1

There was no significant difference in total serum IgM and total serum IgG among conventional 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice and 3H9-µ and V{kappa}8-{kappa} Tg and Tg– littermates (p > 0.05; Fig. 1, A and C); however, little to none of the IgM was Tg-encoded IgMa. Most of the serum IgM and all of the IgG in mice that inherited the conventional 3H9-µ Tg, alone or together with V{kappa}8-{kappa} were derived from endogenous IgMb H chain alleles. Tg-encoded IgMa was consistently 50- to 80-fold lower (p < 0.001) than IgMb in both 3H9-µ-V{kappa}8-{kappa} and 3H9-µ Tg BWF1 mice (Fig. 1, A and C). Serum levels of IgMa and IgMb were similar in 3H9-µ-V{kappa}8-{kappa} and 3H9-µ Tg BWF1 mice (p > 0.05). IgMb titers in 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice and the 3H9-µ and V{kappa}8-{kappa} Tg and Tg– littermates were consistently higher than those in NZW sera in keeping with known elevated IgM levels in BWF1 mice. In contrast to 3H9-µ and 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice, virtually all of the serum IgM in conventional Tg VHNP-µ and VHNP-µ-V{kappa}8-{kappa} Tg BWF1 was Tg-encoded IgMa, although total levels of serum IgM were variable and ~10-fold less than IgM levels in Tg– littermates (Fig. 4A). Total serum IgG was reduced 100-fold or more in VHNP-µ-V{kappa}8-{kappa} Tg BWF1 compared with Tg– littermates (Fig. 4C), consistent with the inability of the VHNP-µ Tg to be isotype switched. IgMa anti-DNA was detected in one-half of the VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice and in 40% of the VHNP-µ Tg BWF1 mice by 6 mo of age (Fig. 4B). Neither IgMb anti-DNA nor IgG anti-DNA was detected in VHNP-µ-V{kappa}8-{kappa} or VHNP-µ Tg BWF1 mice (Fig. 4B and data not shown).



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 4. Total serum Ig and anti-DNA in VHNP-µ-V{kappa}8-{kappa} and VHNP-µ, V{kappa}8-{kappa}, and Tg– littermate BWF1 mice. For details, see legend to Fig. 1.

 
Total serum IgM in 3H9R-V{kappa}4R and 3H9R Tg BWF1 mice was less than that in V{kappa}4R Tg and Tg– littermates (p < 0.05; Fig. 3A), and serum levels of Tg-encoded IgMa and endogenously encoded IgMb were similar in 3H9R-V{kappa}4R Tg BWF1 (p > 0.05). This no doubt reflects the ability of VH3H9 to pair with L chains that do not yield an anti-DNA. Serum levels of IgMb were 6-fold higher than IgMa in 3H9R Tg BWF1 mice (p < 0.01). The different VH expressed as IgMb could be expected to yield a lower percentage of DNA-reactive B cells and be less susceptible to inactivation during development. Total IgG was similar in 3H9R-V{kappa}4R, 3H9R, and V{kappa}4R Tg BWF1 mice and Tg– littermates (Fig. 3C) although IgG encoded by the knock-in Tg allele could not be distinguished from that encoded by an endogenous allele.

Flow cytometric analysis of bone marrow and splenic B cells in Tg mice

There was no statistically significant difference (p > 0.05) in the percentage of B cells identified as surface µ+ cells in 3H9-µ-V{kappa}8-{kappa}, 3H9R-V{kappa}4R, VHNP-µ-V{kappa}8-{kappa}, 3H9R, V{kappa}8-{kappa}, and V{kappa}4R Tg and Tg– BWF1 spleens. The percentage of B cells in 3H9-µ Tg BWF1 was reduced by 40% compared with Tg– mice (p < 0.01). Approximately 85% of splenic B cells in 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice expressed surface IgM derived from the Tg-encoded H chain, 8% expressed surface IgM derived from rearrangements of endogenous H chain alleles, and a small percentage expressed both IgMa and IgMb (Fig. 5B). The percentages of IgMb+ and IgMa+IgMb+ splenic B cells increased with age. 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice >=10 mo old often had >20% IgMb+ and ~12–15% IgMa+IgMb+ B cells (not shown), but 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice 6 mo old had <=1% IgMb+ and <=1% IgMa+IgMb+ B cells (not shown). Surface IgM expression was 2- to 3-fold reduced on IgMa B cells compared with IgMb B cells in 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice or B cells in V{kappa}8-{kappa} Tg and Tg– BWF1 mice (Fig. 6). The narrow range of reduced surface IgMa expression on B cells from 3H9-µ-V{kappa}8-{kappa} Tg BWF1 would be consistent with expression of a monospecific, self-reactive receptor on the majority of B cells (Fig. 5Bb). These results also suggest that relatively few of the 3H9-µ-V{kappa}8-{kappa} B cells were able to eliminate or edit the DNA reactivity of the 3H9-µ-V{kappa}8-{kappa} Tg receptor. Most splenic B cells from VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice expressed relatively high levels of Tg-encoded surface IgMa, consistent with expression of a monospecific, nonautoreactive receptor (Fig. 7Bc). Unlike B cells from 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice (Fig. 5Bd), there were almost no IgMb-expressing B cells in VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice (Figs. 7Bd). Surface IgM expression on splenic B cells from VHNP-µ Tg mice was similar to that for B cells from VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice (not shown).



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 5. Flow cytometric analysis of IgMa and IgMb expression on bone marrow and splenic B cells from 3H9-µ-V{kappa}8-{kappa} (A and B) and age-matched littermate Tg– (C and D) BWF1 mice. Cells from bone marrow and spleen were gated on B220/CD45R+ mononuclear cells. a–d, Percentages of cells. The results are representative of at least three adult mice 3–9 mo old.

 


View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 6. Relative levels of surface IgMa vs IgMb on splenic B cells from representative 3H9-µ-V{kappa}8-{kappa} and V{kappa}8-{kappa} and Tg– littermate BWF1 mice. Splenic B cells from 9-mo-old 3H9-µ-V{kappa}8-{kappa} BWF1 mice were stained with anti-IgMa (dotted line) or anti-IgM (solid line). Splenic B cells from a Tg– BWF1 were stained with anti-IgM (heavy solid line). The anti-IgM reagent binds both IgMa and IgMb. The results are representative of mice 3–9 mo old.

 


View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 7. Flow cytometric analysis of IgMa and IgMb expression on mice bone marrow (A) and splenic (B) B cells from a VHNP-µ-V{kappa}8-{kappa} mice. Cells from both bone marrow and spleen were gated on B220CD45R+ mononuclear cells. a–d, Percentages of cells. The results are representative of two adult mice 3–6 mo old.

 
Surface IgMa expression on B cells from 3H9R-V{kappa}4R, 3H9R (not shown) and 3H9-µ (Fig. 8) Tg BWF1 mice was different from that on B cells from both VHNP-µ-V{kappa}8-{kappa}8 and 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice. Surface IgMa expressions on B cells from 3H9R-V{kappa}4R (Fig. 9) and 3H9R (not shown) were identical. Surface IgMa+ splenic B cells from 3H9R-V{kappa}4R, 3H9R, and 3H9-µ Tg BWF1 mice formed three distinct subpopulations (Figs. 8 and 9B). Surface IgMa expression on the c subpopulation of splenic B cells was similar to surface IgMa expression on the majority of B cells in VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice and the highest level of expression on B cells in Tg– BWF1 (Fig. 9Bc). The b subpopulation of splenic IgMa B cells in 3H9R-V{kappa}4R, 3H9R, and 3H9-µ Tg BWF1 mice had surface IgMa expression similar to that for splenic B cells from 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice and 2- to 3-fold less than that for B cells from VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice (Fig. 7Bb vs Figs. 8b and 9Bb). Surface IgMa expression on the "a" subpopulation of splenic B cells in 3H9R-V{kappa}4R, 3H9R, and 3H9-µ Tg BWF1 was even more reduced, 10-fold or more compared with surface IgMa expression on VHNP-µ-V{kappa}8-{kappa} splenic B cells (Fig. 7Bc vs 8a and 9Ba). The percentage of IgMb+ splenic B cells in 3H9R-V{kappa}4R Tg BWF1 was approximately one-fourth that in 3H9-µ-V{kappa}8-{kappa} Tg BWF1. L chain expression in both site-directed 3H9R and conventional 3H9-µ B cells was determined by rearranged endogenous alleles. L chains that vetoed the DNA specificity of 3H9R or 3H9-µ (35, 36) would be expected to yield the B cells detected in subpopulation c (Figs. 8 and 9B). Subpopulation "c" in 3H9R and 3H9R-V{kappa}4R Tg BWF1 mice would also contain B cells that edited their DNA-reactive IgMa receptors to eliminate autoreactivity. IgMa B cells with L chains that could not effectively veto the specificity of 3H9R or 3H9-µ would be expected to be in subpopulations a and b. The difference between the a and b subpopulations probably relates to DNA reactivity with the a subpopulation having higher relative affinity for DNA, such as with VH3H9-V{kappa}4. B cells in the b subpopulation are more likely to have lower relative affinity for DNA, such as with VH3H9-V{kappa}8. As expected, IgMa B cells from 3H9R and 3H9R-V{kappa}4R Tg BWF1 mice also expressed surface IgD, but IgMa B cells from 3H9-µ, 3H9-µ-V{kappa}8-{kappa}, VHNP-µ, and VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice did not have surface IgD (not shown).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 8. Flow cytometric analysis of IgMa and IgMb expression on splenic B cells from 3H9-µ BWF1 mice. Cells were gated on B220/CD45R+ mononuclear cells. a–d, Percentages of cells. The results are representative of at least three adult mice 3–7 mo old.

 


View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 9. Flow cytometric analysis of IgMa and IgMb expression on bone marrow and splenic B cells from 3H9R-V{kappa}4R (A and B) and age-matched littermate Tg– (C and D) BWF1 mice. Cells from both bone marrow and spleen were gated on B220/CD45R+ mononuclear cells. a–d, Percentages of cells. The results are representative of at least three adult mice 3–7 mo old.

 
The reduced surface IgM expression on IgMa splenic B cells in 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice was also apparent in bone marrow (Fig. 5A), and the percentage of B220/CD45R+ surface IgM+ cells in bone marrow from 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice was much higher than the percentage of B220/CD45R+, surface IgM+ cells in either VHNP-µ-V{kappa}8-{kappa} Tg or non-Tg BWF1 mice. More than 90% of B220/CD45R+ bone marrow cells were surface IgMa+ vs 80% IgMa+ in VHNP-µ-V{kappa}8-{kappa} Tg (Fig. 7A) and only 39% IgMb+ in non-Tg BWF1 (Fig. 5C). Similarly to splenic B cells, surface IgMa expression on bone marrow B cells in 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice was also reduced compared with bone marrow B cells in VHNP-µ-V{kappa}8-{kappa}8 and non-Tg BWF1 mice. These results are consistent with the hypothesis that 3H9-µ-V{kappa}8-{kappa} B cells encountered autoantigen in the bone marrow and were arrested in their development. Surface expression of IgMa on bone marrow B cells in 3H9R-V{kappa}4R Tg BWF1 mice was reduced compared with IgM expression on non-Tg B cells (Fig. 5C and Fig. 9, A and C) and IgMa expression on B cells from VHNP-µ-V{kappa}8-{kappa} BWF1 mice (Fig. 7A). The percentage of IgM+, B220/CD45R+ was similar for bone marrow from 3H9R-V{kappa}4R and non-Tg BWF1 mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Anti-DNA Tg BWF1 mice were used to analyze DNA-specific B cell development and activation in an autoimmune background. Mice monospecific for an anti-DNA Ab formed by the 3H9VH and either V{kappa}8 or V{kappa}4 were compared with mice that inherited only 3H9VH, V{kappa}8, or V{kappa}4 or were Tg–. Mice inherited the 3H9VH as either a conventional 3H9VH-µ Tg or the 3H9RVH knock-in Tg targeted to JH. V{kappa}8 was inherited as a conventional V{kappa}8-{kappa} Tg, and V{kappa}4, as the V{kappa}4R V{kappa} knock-in Tg targeted to J{kappa}. The results indicate that B cell development and autoantibody production differ remarkably in BWF1 mice that inherited the conventional 3H9-µ H chain compared with the 3H9R VH knock-in. When B cells expressed VH3H9 as a knock-in Tg, the respective BWF1 mice developed autoimmune anti-DNA similarly to Tg– BWF1 mice. When B cells expressed VH3H9 as a conventional Tg, only a small percentage of the respective BWF1 mice produced relatively low serum anti-DNA titers detected only late in life compared with Tg– BWF1 mice. Most serum IgM and all of the IgM anti-DNA produced in the conventional Tg BWF1 mice was IgMb derived from endogenous V gene rearrangements rather than the VH3H9-µ Tg. In contrast, much of the serum IgM and most of the IgM anti-DNA produced in the site-directed Tg BWF1 mice were products of the 3H9R Tg. We expect that the IgG produced in site-directed 3H9R Tg BWF1 mice was also encoded by the 3H9R Tg. The majority of B cells expressing VH3H9 as either the conventional or knock-in Tg had a phenotype consistent with Ag exposure during development in the bone marrow and subsequent inactivation and anergy. All of the IgM produced in BWF1 mice that inherited the conventional VHNP-µ Tg with or without the V{kappa}8-{kappa} Tg was Tg IgMa. B cells in the VHNP-µ Tg BWF1 mice did not have an anergic cell surface phenotype. Overall, the results indicate that central tolerance is effective in BWF1 and that developmental arrest of IgMa B cells was sustained in the periphery in the conventional 3H9-µ but not knock-in 3H9R Tg BWF1 mice.

Several functional differences between the conventional 3H9-µ and the knock-in 3H9R Tgs could contribute to the phenotypic differences between 3H9-µ and 3H9R Tg BWF1 mice and the respective 3H9-µ-V{kappa}8-{kappa} and 3H9R-V{kappa}4R double Tg BWF1 mice. 1) The 3H9R and V{kappa}4R Tgs can be edited (22, 26); however, the 3H9-µ and V{kappa}8-{kappa} Tgs cannot. 2) Mature IgMa B cells in 3H9R, but not 3H9-µ BWF1, express surface IgD. 3) Mature IgMa B cells in 3H9R but not 3H9-µ BWF1 mice can switch the Tg VH3H9 to IgG or other constant regions. The potential for both 3H9R and V{kappa}4R to be edited may explain to some extent why the knock-in Tg BWF1 mice had higher levels of serum IgMa than those detected in the sera of conventional Tg BWF1 mice. The difference in editing potential between conventional and site-directed Tgs is less likely to explain why 3H9R Tg BWF1 mice produce IgMa, including IgMa anti-DNA, but 3H9-µ Tg BWF1 mice did not. The Tg H chains in both 3H9-µ and 3H9R Tg B cells could be paired with any L chain, and surface IgMa expression was similar on B cells from 3H9-µ and 3H9R Tg BWF1 mice. These results suggest that the difference in production of Tg-encoded IgMa and IgMa anti-DNA between 3H9-µ and 3H9R Tg BWF1 mice was more likely determined by the potential for activation than by susceptibility to induction of anergy, particularly for IgMa B cells in the c subpopulation (Fig. 8). Potential Ags for IgMa B cells should be no different between 3H9-µ and 3H9R Tg BWF1 mice; however, IgMa B cells in 3H9R but not 3H9-µ Tg BWF1 mice expressed IgD and could switch VH3H9 or a different VH generated by editing on the Tg allele to IgG or other constant regions. Recently reported results have indicated that IgD may be important for B cell activation and germinal center formation (37, 38). Likewise, signaling through B cell surface IgG is more effective for signaling activation, and consequently, the induction of more clonal expansion than signaling through IgM (39). An inability to class switch should not prevent activation to IgM production, however, and does not explain why 3H9-µ Tg BWF1 mice produced little IgMa and no IgMa anti-DNA. Even though IgMa B cells did not appear to be anergic in either VHNP-µ or VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice, total serum IgM was markedly reduced in both compared with Tg– BWF1 mice. Reduced serum IgM in VHNP-µ Tg BWF1 mice was most likely due to limited B cell activation and IgM Ab production caused by the absence of an appropriate immune stimulus, the absence of IgD, or both. The VHNP-µ Tg did not include C{delta} or C{gamma}. Assuming that attempts to edit the VHNP-µ Tg would have resulted in its elimination and IgMb production, there appeared to be little attempt to edit the Tg H chain in VHNP-µ-V{kappa}8-{kappa} and VHNP-µ Tg BWF1 mice, probably because the respective receptors did not have autoimmune specificity. The IgMa anti-DNA detected in some of the VHNP-µ and VHNP-µ-V{kappa}8-{kappa} Tg BWF1 mice was most likely produced by B cells expressing a VL other than V{kappa}8, yielding an anti-DNA that did not induce anergy. Analyses of IgMa and IgMb B cells sorted by flow cytometry and IgMa- and IgMb-producing hybridomas derived from 3H9-µ and 3H9R BWF1 mice and IgMa-producing hybridomas from VHNP-µ and VHNP-µ-V{kappa}8-{kappa} Tg BWF1 are currently under way. The results will provide insight about the effects of the VHNP-µ, 3H9-µ, and 3H9R Tgs on the functional status of B cells in Tg BWF1 mice.

The results from studies of BWF1 mice Tg for the conventional 3-32-µ anti-DNA H chain are consistent with the results and conclusion described above for 3H9-µ (31). 3-32-µ BWF1 mice also had very low serum levels of Tg-encoded IgMa and IgMa anti-DNA titers no higher than those detected in normal BALB/c mice. The 3-32-µ Tg also did not include C{delta} and could not be switched to C{gamma}. B cells in R4A-{gamma}2b conventional Tg BWF1, in contrast, were not anergic and produced Tg-encoded serum anti-DNA at higher titers than in normal, non-Tg BWF1 mice (30). Although it is not clear why expression of the R4A-{gamma}2b Tg in BWF1 mice would have a different influence on B cell development and activation than 3H9-µ or 3-32-µ, the effect may be due to the effects of expression of C{gamma} vs Cµ during B cell development (40). The more effective activation signaling through C{gamma} (39) noted above may determine the difference in anti-DNA production from the R4A-{gamma}2b vs 3H9-µ or 3-32-µ Tgs. Alternatively, differences in specificity between R4A-{gamma}2b and 3H9-µ- or 3-32-µ-encoded Abs could have contributed to differences in B cell development and activation in R4A-{gamma}2b vs 3H9-µ or 3-32-µ Tg BWF1 mice. BWF1 mice Tg for the D42 anti-DNA VH knock-in Tg (32) had a phenotype similar to that of 3H9R-V{kappa}4R and 3H9R knock-in Tg BWF1 mice. D42 VH knock-in BWF1 mice produced anti-DNA encoded by the Tg. The D42 VH knock-in Tg could be expressed with C{delta} and isotype switched to C{gamma} or other CH similarly to the 3H9R knock-in Tg. 3H9-µ and VHNP-µ Tg BWF1 mice are similar in many respects to 2–12H Tg C.B-17 mice (41) although C.B-17 mice are not autoimmune prone. The 2-12H Tg is a conventional µ, anti-Sm Tg H chain. 2-12H Tg mice produce very little Tg serum IgM, whether specific for Sm or not, even though 2-12H expressing B cells seem to be fully functional. In 2-12H Tg mice in which B cell receptor signaling was enhanced by a human CD19 Tg, serum IgM production was restored to levels greater than that in normal, non-Tg mice (42). The absence of 2-12H Tg-encoded Ab is thought to be due to clonal ignorance and differentiation of 2-12H Tg B cells to B-1 which have a higher signaling threshold for activation.

Because there were IgMb B cells that did not express IgMa in 3H9-µ-V{kappa}8-{kappa} and 3H9-µ Tg BWF1 mice, the 3H9-µ Tg must have been inactivated or eliminated in the respective B cells, likely as an attempt to edit autoreactivity. Assuming that rearrangements of endogenous H and L chain alleles are random, rearrangement of endogenous H and/or L chain alleles should have included VH and VL combinations that encoded anti-DNA. Yet, fewer than 10% of 3H9-µ-V{kappa}8-{kappa} BWF1 mice and 25% of 3H9-µ BWF1 mice produced detectable IgM or IgG anti-DNA when 1 year old or less. B cells from 3H9-µ-V{kappa}8-{kappa} Tg BWF1 spleen and bone marrow also included a subpopulation of B cells that expressed both IgMa and IgMb on the cell surface. If rearrangement and expression of an endogenous H chain allele generated an IgMb that bound DNA, the respective B cell may have expressed, at least temporarily, two cell surface anti-DNA antibodies, likely with different epitope specificity. Expression of two different anti-DNA Abs could in turn have yielded even stronger receptor cross-linking on the respective B cells and thus stronger signaling leading to deletion or more profound anergy of the respective B cells in the bone marrow. IgMb Abs that did not bind DNA or other relevant autoantigens could have diluted the negative signaling resulting in survival and escape from anergy, especially if the Tg IgMa loci in the respective B cells were inactivated or eliminated. In fact, µ expression from a rearranged endogenous allele could have disrupted the DNA specificity of 3H9-µ Ab receptors. Likewise, expression of L chains that vetoed DNA reactivity would have also disrupted the DNA specificity of 3H9-µ Ab receptors. Expression of both 3H9-µ and µ derived from an endogenous rearrangement was detected in many hybridomas from 3H9-µ Tg MRLlpr/lpr (43). Secreted IgM from hybridomas that expressed both 3H9-µ and µ from an endogenous allele either did not bind to DNA or did so with an ANA pattern different from 3H9. The relatively small amount of IgMa detected in sera of 3H9-µ-V{kappa}8-{kappa} and 3H9-µ Tg BWF1 mice could have been produced by B cells expressing both IgMa and IgMb. Because either or both H and L loci containing 3H9R and V{kappa}4R, respectively, could be edited to eliminate and/or disrupt DNA specificity, IgMa B cells in 3H9R-V{kappa}4R and 3H9R Tg BWF1 mice would have had more opportunity to generate non DNA-reactive receptors. B cells in 3H9R-V{kappa}4R and 3H9R Tg BWF1 that edited either 3H9R or V{kappa}4R to generate another anti-DNA Ab would also have expressed two different anti-DNA antibodies. Such B cells would have been susceptible to the same toleragenic signaling as B cells in 3H9-µ-V{kappa}8-{kappa} and 3H9-µ BWF1 mice. The two subpopulations of 3H9R-V{kappa}4R splenic B cells with low and very low level expression of surface IgMa (Fig. 9B, a and b) likely included B cells that either did not edit the high affinity 3H9-V{kappa}4 receptor or those that did edit the receptor but generated another anti-DNA or other autoreactive receptor. The production of IgMb anti-DNA in 3H9R-V{kappa}4R and 3H9R Tg BWF1 mice is evidence that editing could lead to generation of a second anti-DNA receptor; however, IgMb anti-DNA production was generally only 12% or less of the IgMa anti-DNA in the same mice. Recent results obtained from 3H9 and 3H9/56R knock-in Tg MRLlpr/lpr mice indicate that V gene editing can generate B cells that express two different cell surface receptors (44, 45). When only one of the receptors was specific for DNA, the respective B cells were rescued from anergy or deletion. The IgMa and IgG anti-DNA in 3H9R-V{kappa}4R and 3H9R Tg BWF1 mice may have been produced by 3H9R Tg B cells that escaped tolerance induction or by B cells in which receptor editing generated new anti-DNA receptors that did not induce tolerance.

Raff et al. (46) were the first to describe the effects of Ig receptor engagement on immature bone marrow B cells. Prolonged exposure of immature B cells to anti-Ig in vitro resulted in the irreversible inactivation of the respective B cells. The effects of Ig receptor engagement on immature bone marrow B cells are similar when the engagement is by specific Ag in vivo (16, 18). When non-autoimmune-prone mice were made Tg for production of soluble lysozyme (hen egg lysozyme; HEL) and IgM and IgD that bind lysozyme with high affinity (HEL-Ig), B cells in the double-Tg mice were anergic (18). HEL-specific B cells in mice Tg for HEL and for the same high affinity anti-HEL as a knock-in VH with the conventional VL were also anergic (47). Anergy of HEL-specific B cells was dependent on the presence of soluble HEL, and as long as soluble HEL was present, T cell help could not stimulate B cell differentiation to Ab production (48). HEL-specific B cells were excluded from follicles in HEL-Tg mice (49). The greatly increased percentage of B cells with reduced IgMa expression in the bone marrow of conventional 3H9-µ and knock-in 3H9R Tg BWF1 mice, with or without V{kappa}8-{kappa} or V{kappa}4R, respectively, indicates that immature, DNA-specific B cells must have encountered relevant Ag in the bone marrow and were developmentally arrested. DNA-specific B cells were also developmentally arrested in 3H9-µ Tg BALB/c and MRL+/+ mice (19, 50), and like 3H9-µ Tg BWF1 mice, the developmental arrest was sustained in the periphery. Developmental arrest of DNA-reactive B cells was not sustained in 3H9-µ Tg MRLlpr/lpr (50) and BALB/clpr/lpr (51) mice. When anergic, DNA-specific B cells in 3H9-µ Tg BALB/c mice were provided a source of T cell help, the developmentally arrested, DNA-specific B cells not only entered follicles but were also activated to produce anti-DNA Ab (51). Follicular inclusion of DNA-reactive B cells and anti-DNA autoantibody production in 3H9-µ Tg MRLlpr/lpr (50) and BALB/clpr/lpr (52) mice were also Th cell dependent (51). When CD4+CD25+ T-regulatory cells were provided along with Th cells, DNA-specific B cells were still follicularly included but did not differentiate to anti-DNA Ab-producing cells. Even though anergic B cells may affect the development of activated Th cells in BWF1 mice (53), reduced T cell help or a difference in CD4+CD25+ T-regulatory cells seems unlikely to account for the difference in IgMa B cell activation between 3H9-µ and 3H9R Tg BWF1 mice. 3H9-µ and 3H9R Tg BWF1 had similar percentages of anergic B cells and similar total serum IgG, and both 3H9-µ and 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice produced IgG ANA. What is unknown at present is whether 3H9-µ and 3H9R Tg B cells differ in their potential for Ag presentation to Th cells. Experiments are under way to determine whether anti-DNA B cells in 3H9-µ and 3H9R Tg BWF1 mice are follicularly included and how 3H9-µ and 3H9R Tg B cells may differ in their ability to present Ag to Th cells and respond to T cell help.

In the end, tolerance to DNA in 3H9-µ and 3H9-µ-V{kappa}8-{kappa} Tg BWF1 mice must be due to the inability of the autoimmune stimulation normally present in the lymphoid periphery of BWF1 mice to achieve the threshold of activation needed to overcome the developmental arrest imposed on 3H9-µ Tg-expressing B cells in the bone marrow. A combination of factors including but not limited to the specificity of 3H9, the inability of 3H9-µ and V{kappa}8-{kappa} to be edited, the lack of IgD on B cells expressing only IgMa, the inability of 3H9-µ to be isotype switched, and reduced T cell help as a result of the large number of anergic, DNA-specific B cells may contribute to the sustained B cell tolerance for DNA in conventional Tg 3H9-µ and 3H9-µ-V{kappa}8-{kappa} BWF1. Unlike self-reactive B cells in transgenic, non-autoimmune-prone mice that remain tolerant to self Ag, DNA-reactive B cells in 3H9R and 3H9R-V{kappa}4R Tg BWF1 mice can be activated to overcome the anergy imposed on them during development and produce autoantibody.


   Acknowledgments
 
We thank R. Bray Williams for critical participation in the early stages of this research, Dr. Garnett Kelsoe for C.B-17 VHH50G Tg mice, Dr. Jan Erikson for 3H9-µ and V{kappa}8-{kappa} Tg BALB/c mice, and Dr. Martin Weigert for 3H9R and V{kappa}4R Tg BALB/c mice. We also acknowledge Dr. Richard Cross at St. Jude Children’s Hospital for expert assistance with flow cytometry experiments and Dr. Marko Radic for helpful discussion and critical reading of the manuscript.


   Footnotes
 
1 This research was supported by National Institutes of Health Grant AI26833. Back

2 Address correspondence and reprint requests to Dr. Tony N. Marion, Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163. E-mail address: tmarion{at}utmem.edu Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; Tg, transgene or transgenic; ANA, anti-nuclear Ab; NP, (4-hydroxy-3-nitrophenyl)acetyl; HEL, hen egg lysozyme. Back

Received for publication August 6, 2003. Accepted for publication March 22, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tan, E. M., A. S. Cohen, J. F. Fries, A. T. Masi, D. J. McShane, N. F. Rothfield, J. G. Schaller, N. Talal, R. J. Winchester. 1982. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25:1271.[Medline]
  2. Tan, E. M.. 1989. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv. Immunol. 44:93.[Medline]
  3. Winfield, J. B., I. Faiferman, D. Koffler. 1977. Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus: Association of high avidity anti-native DNA antibody with glomerulonephritis. J. Clin. Invest. 59:90.
  4. Andres, G. A., B. F. Erlanger, K. C. Hsu, B. C. Seegal. 1970. Localization of fluorescein-labeled anti-nucleoside antibodies in glomeruli of patients with active systemic lupus erythematosus nephritis. J. Clin. Invest. 49:2106.
  5. Howie, J. B., B. J. Heyler. 1968. The immunology and pathology of NZB mice. Adv. Immunol. 9:215.[Medline]
  6. Andrews, B. S., R. A. Eisenberg, A. N. Theofilopoulos, S. Izui, C. B. Wilson, P. J. McConahey, E. D. Murphy, J. B. Roths, F. J. Dixon. 1978. Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J. Exp. Med. 148:1198.[Abstract/Free Full Text]
  7. Ternynck, T., S. Avrameas. 1986. Murine natural monoclonal autoantibodies: a study of their polyspecificities and their affinities. Immunol. Rev. 94:99.[Medline]
  8. Izui, S., T. Kobayakawa, M. J. Zyrd, J. Louis, P. H. Lambert. 1977. Mechanism for induction of anti-DNA antibodies by bacterial lipopolysaccharides in mice. II. Correlation between anti-DNA induction and polyclonal antibody formation by various polyclonal B lymphocyte activators. J. Immunol. 119:2157.[Abstract/Free Full Text]
  9. Steward, M. W., F. C. Hay. 1976. Changes in immunoglobulin class and subclass on anti-DNA antibodies with increasing age in NZB/W F1 hybrid mice. Clin. Exp. Immunol. 26:363.[Medline]
  10. Papoian, R., R. Pillarisetty, N. Talal. 1977. Immunological regulation of spontaneous antibodies to DNA and RNA. II. Sequential switch from IgM to IgG in NZB/NZW F1 mice. Immunol. 32:75.[Medline]
  11. Tillman, D. M., N. T. Jou, R. J. Hill, T. N. Marion. 1992. Both IgM and IgG anti-DNA antibodies are the products of clonally selective B cell stimulation in (NZB x NZW)F1 mice. J. Exp. Med. 176:761.[Abstract/Free Full Text]
  12. Cocca, B. A., A. M. Cline, M. Z. Radic. 2002. Blebs and apoptotic bodies are B cell autoantigens. J. Immunol. 169:159.[Abstract/Free Full Text]
  13. Desai, D. D., M. R. Krishnan, J. T. Swindle, T. N. Marion. 1993. Antigen-specific induction of antibodies against native mammalian DNA in nonautoimmune mice. J. Immunol. 151:1614.[Abstract]
  14. Rekvig, O. P., K. Fredriksen, B. Brannsether, U. Moens, A. Sundsfjord, T. Traavik. 1992. Antibodies to eukaryotic, including autologous, native DNA are produced during BK virus infection, but not after immunization with non-infectious BK DNA. Scand. J. Immunol. 36:487.[Medline]
  15. Desai, D. D., T. N. Marion. 2000. Induction of anti-DNA antibody with DNA-peptide complexes. Int. Immunol. 12:1569.[Abstract/Free Full Text]
  16. Nemazee, D. A., K. Burki. 1989. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature 337:562.[Medline]
  17. Hartley, S. B., J. Crosbie, R. Brink, A. B. Kantor, A. Basten, C. C. Goodnow. 1991. Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens. Nature 353:765.[Medline]
  18. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676.[Medline]
  19. Erikson, J., M. Z. Radic, S. A. Camper, R. R. Hardy, C. Carmack, M. Weigert. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349:331.[Medline]
  20. Tiegs, S. L., D. M. Russell, D. Nemazee. 1993. Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177:1009.[Abstract/Free Full Text]
  21. Radic, M. Z., J. Erikson, S. Litwin, M. Weigert. 1993. B lymphocytes may escape tolerance by revising their antigen receptors. J. Exp. Med. 177:1165.[Abstract/Free Full Text]
  22. Prak, E. L., M. Weigert. 1995. Light chain replacement: a new model for antibody gene rearrangement. J. Exp. Med. 182:541.[Abstract/Free Full Text]
  23. Chen, C., Z. Nagy, M. Z. Radic, R. R. Hardy, D. Huszar, S. A. Camper, M. Weigert. 1995. The site and stage of anti-DNA B-cell deletion. Nature 373:252.[Medline]
  24. Shlomchik, M. J., A. H. Aucoin, D. S. Pisetsky, M. G. Weigert. 1987. Structure and function of anti-DNA autoantibodies derived from a single autoimmune mouse. Proc. Natl. Acad. Sci. USA 84:9150.[Abstract/Free Full Text]
  25. Dal Porto, J. M., A. M. Haberman, M. J. Shlomchik, G. Kelsoe. 1998. Antigen drives very low affinity B cells to become plasmacytes and enter germinal centers. J. Immunol. 161:5373.[Abstract/Free Full Text]
  26. Chen, C., Z. Nagy, E. L. Prak, M. Weigert. 1995. Immunoglobulin heavy chain gene replacement: a mechanism of receptor editing. Immunity 3:747.[Medline]
  27. Dal Porto, J. M., A. M. Haberman, G. Kelsoe, M. J. Shlomchik. 2002. Very low affinity B cells form germinal centers, become memory B cells, and participate in secondary immune responses when higher affinity competition is reduced. J. Exp. Med. 195:1215.[Abstract/Free Full Text]
  28. Carmack, C. E., S. A. Camper, J. J. Mackle, W. U. Gerhard, M. G. Weigert. 1991. Influence of a V{kappa}8 L chain transgene on endogenous rearrangements and the immune response to the HA(Sb) determinant on influenza virus. J. Immunol. 147:2024.[Abstract]
  29. Radic, M. Z., M. A. Mascelli, J. Erikson, H. Shan, M. Weigert. 1991. Ig H and L chain contributions to autoimmune specificities. J. Immunol. 146:176.[Abstract]
  30. Spatz, L., V. Saenko, A. Iliev, L. Jones, L. Geskin, B. Diamond. 1997. Light chain usage in anti-double-stranded DNA B cell subsets: role in cell fate determination. J. Exp. Med. 185:1317.[Abstract/Free Full Text]
  31. Wellmann, U., A. Werner, T. H. Winkler. 2001. Altered selection processes of B lymphocytes in autoimmune NZB/W mice, despite intact central tolerance against DNA. Eur. J. Immunol. 31:2800.[Medline]
  32. Friedmann, D., N. Yachimovich, G. Mostoslavsky, Y. Pewzner-Jung, A. Ben-Yehuda, K. Rajewsky, D. Eilat. 1999. Production of high affinity autoantibodies in autoimmune New Zealand Black/New Zealand White F1 mice targeted with an anti-DNA heavy chain. J. Immunol. 162:4406.[Abstract/Free Full Text]
  33. Chen, C., E. L. Prak, M. Weigert. 1997. Editing disease-associated autoantibodies. Immunity 6:97.[Medline]
  34. Marion, T. N., A. R. Lawton, J. F. Kearney, D. E. Briles. 1982. Anti-DNA autoantibodies in (NZB x NZW)F1 mice are clonally heterogeneous, but the majority share a common idiotype. J. Immunol. 128:668.[Abstract]
  35. Ibrahim, S. M., M. Weigert, C. Basu, J. Erikson, M. Z. Radic. 1995. Light chain contribution to specificity in anti-DNA antibodies. J. Immunol. 155:3223.[Abstract]
  36. Prak, E. L., M. Trounstine, D. Huszar, M. Weigert. 1994. Light chain editing in {kappa}-deficient animals: a potential mechanism of B cell tolerance. J. Exp. Med. 180:1805.[Abstract/Free Full Text]
  37. Kim, K. M., M. Reth. 1995. The B cell antigen receptor of class IgD induces a stronger and more prolonged protein tyrosine phosphorylation than that of class IgM. J. Exp. Med. 181:1005.[Abstract/Free Full Text]
  38. Chaturvedi, A., Z. Siddiqui, F. Bayiroglu, K. V. Rao. 2002. A GPI-linked isoform of the IgD receptor regulates resting B cell activation. Nat. Immunol. 3:951.[Medline]
  39. Wakabayashi, C., T. Adachi, J. Wienands, T. Tsubata. 2002. A distinct signaling pathway used by the IgG-containing B cell antigen receptor. Science 298:2392.[Abstract/Free Full Text]
  40. Kurtz, B. S., P. L. Witte, U. Storb. 1997. {gamma}2b provides only some of the signals normally given via mu in B cell development. Int. Immunol. 9:415.[Abstract/Free Full Text]
  41. Santulli-Marotto, S., M. W. Retter, R. Gee, M. J. Mamula, S. H. Clarke. 1998. Autoreactive B cell regulation: peripheral induction of developmental arrest by lupus-associated autoantigens. Immunity 8:209.[Medline]
  42. Qian, Y., C. Santiago, M. Borrero, T. F. Tedder, S. H. Clarke. 2001. Lupus-specific anti-ribonucleoprotein B cell tolerance in nonautoimmune mice is maintained by differentiation to B-1 and governed by B cell receptor signaling thresholds. J. Immunol. 166:2412.[Abstract/Free Full Text]
  43. Roark, J. H., C. L. Kuntz, K. A. Nguyen, L. Mandik, M. Cattermole, J. Erikson. 1995. B cell selection and allelic exclusion of an anti-DNA Ig transgene in MRLlpr/lpr mice. J. Immunol. 154:4444.[Abstract]
  44. Li, Y., H. Li, D. Ni, M. Weigert. 2002. Anti-DNA B cells in MRL/lpr mice show altered differentiation and editing pattern. J. Exp. Med. 196:1543.[Abstract/Free Full Text]
  45. Li, Y., H. Li, M. Weigert. 2002. Autoreactive B cells in the marginal zone that express dual receptors. J. Exp. Med. 195:181.[Abstract/Free Full Text]
  46. Raff, M. C., J. J. Owen, M. D. Cooper, A. R. Lawton, M. Megson, W. E. Gathings. 1975. Differences in susceptibility of mature and immature mouse B lymphocytes to anti-immunoglobulin-induced immunoglobulin suppression in vitro. Possible implications for B-cell tolerance to self. J. Exp. Med. 142:1052.[Abstract/Free Full Text]
  47. Phan, T. G., M. Amesbury, S. Gardam, J. Crosbie, J. Hasbold, P. D. Hodgkin, A. Basten, R. Brink. 2003. B cell receptor-independent stimuli trigger immunoglobulin (Ig) class switch recombination and production of IgG autoantibodies by anergic self-reactive B cells. J. Exp. Med. 197:845.[Abstract/Free Full Text]
  48. Goodnow, C. C., R. Brink, E. Adams. 1991. Breakdown of self-tolerance in anergic B lymphocytes. Nature 352:532.[Medline]
  49. Cyster, J. G., S. B. Hartley, C. C. Goodnow. 1994. Competition for follicular niches excludes self-reactive cells from the recirculating B cell repertoire. Nature 371:389.[Medline]
  50. Mandik-Nayak, L., S. J. Seo, C. Sokol, K. M. Potts, A. Bui, J. Erikson. 1999. MRLlpr/lpr mice exhibit a defect in maintaining developmental arrest and follicular exclusion of anti-double-stranded DNA B cells. J. Exp. Med. 189:1799.[Abstract/Free Full Text]
  51. Seo, S. J., M. L. Fields, J. L. Buckler, A. J. Reed, L. Mandik-Nayak, S. A. Nish, R. J. Noelle, L. A. Turka, F. D. Finkelman, A. J. Caton, J. Erikson. 2002. The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity 16:535.[Medline]
  52. Fields, M. L., C. L. Sokol, A. Eaton-Bassiri, S. Seo, M. P. Madaio, J. Erikson. 2001. Fas/fas ligand deficiency results in altered localization of anti-double-stranded DNA B cells and dendritic cells. J. Immunol. 167:2370.[Abstract/Free Full Text]
  53. Wellmann, U., M. Letz, A. Schneider, K. Amann, T. H. Winkler. 2001. An Ig mu-heavy chain transgene inhibits systemic lupus erythematosus immunopathology in autoimmune (NZB x NZW)F1 mice. Int. Immunol. 13:1461.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 LupusHome page
A Guth, T Detanico, D Smith, K. Tung, C Bonorino, and L. Wysocki
Spontaneous autoimmunity in mice that carry an IghV partial transgene: a required arginine in VHCDR3
Lupus, April 1, 2009; 18(4): 299 - 308.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steeves, M. A.
Right arrow Articles by Marion, T. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steeves, M. A.
Right arrow Articles by Marion, T. N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS