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Arthritis and Immunology, Oklahoma Medical Research Foundation, and College of Medicine, University of Oklahoma Health Sciences Center and U.S. Department of Veteran’s Affairs Medical Center, Oklahoma City, OK 73104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Spliceosomal proteins, which are bound by anti-Sm and anti-nuclear ribonucleoprotein (nRNP) autoantibodies, are common targets of this abnormal lupus autoimmune response. These Sm and nRNP Ags are subsets of polypeptides associated with U small nuclear RNAs that are involved in the splicing of pre-mRNA. Anti-Sm Abs are found in approximately 20 to 25% of all SLE patient sera. Indeed, these Abs are so specific for SLE in man that they have become one of the classification criteria (1). These anti-Sm autoantibodies are directed predominantly against the Sm B/B' and Sm D proteins (2, 3) and immunoprecipitate the U1, U2, U4/U6, and U5 RNAs (4). In lupus patients as much as 20% of the entire Ig repertoire may bind Sm (5). Lupus patient sera with mature anti-Sm and anti-nRNP precipitin responses identify 11 antigenic regions of B/B' that have been identified by binding to peptides taken from the primary sequence (6).
Nearly all the SLE patients who have anti-Sm Abs also have anti-nRNP Abs. Those who begin with anti-Sm alone virtually always develop anti-nRNP over the course of their disease (7). Anti-nRNP immunoprecipitate only U1 RNA and bind the nRNP 70K, nRNP A, and nRNP C proteins. About 40% of SLE patient sera precipitate the nRNP proteins (8).
Much work has been done with spontaneously arising animal models of lupus (9, 10, 11). Recently, a peptide-induced model of lupus autoimmunity has been established in New Zealand White rabbits by immunization with one antigenic octapeptide of a spliceosomal complex, either PPPGMRPP or PPPGIRGP (5). These peptides, which are repeated four times in Sm B/B', have been previously identified as major antigenic targets of the human SLE response (6). Indeed, autoantibodies binding to these sequences account for up to 40% of the anti-Sm response in certain patients (our unpublished observations). In addition, anti-Sm Abs from all patient sera tested bind these peptides. Somewhat similar serologic results were obtained by Elkon and colleagues (12) with the carboxyl-terminal 22 amino acids. In our hands, two peptides, PPPGMRPP and PPPGIRGP, are the initial targets of the anti-Sm autoimmune response of four patients with the least complicated initial B cell epitopes (5) (our unpublished observations).
After rabbits develop an immune response to the short peptide of immunization, additional boosting is associated with a fully mature humoral autoimmune response common in SLE, which includes precipitating levels of anti-Sm and anti-nRNP, anti-dsDNA Abs, and high titer antinuclear Abs. Clinical features of SLE, including proteinuria, cellular and granular renal casts, hypoalbuminemia, thrombocytopenia, alopecia, and seizures, are also observed in these rabbits.
Although very useful for large scale production of Abs, rabbits are difficult subjects for basic immunologic and genetic analysis. In this study we examine 13 different strains of inbred mice for their capacity to produce lupus autoimmunity after immunization with the PPPGMRPP peptide of Sm B/B'.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strains of 6-wk-old, female, inbred mice with various H-2 associations were obtained from The Jackson Laboratory (Bar Harbor, Maine). These strains include 129/J, A/J, AKR/J, C57BL/6J, C57BL/10J, C57BL/J, C3H/HeJ, DBA/2J, BALB/c Bailey, NZB/BINJ, SJL/J, SWR/J, and PL/J. Mice were kept in a pathogen-free, American Association for Accredidation of Laboratory Animal Care-accredited facility. In addition, the congenic strains, B6.AKR-H-2k/FlaEg and A.BY/SnJ-H-2b, were obtained from The Jackson Laboratory and maintained in the same facility.
Human sera
SLE patient sera with precipitating levels of anti-Sm and/or anti-nRNP autoantibodies were selected as positive controls for various assays from our stored collection of patient serum samples. Control normal sera were also available from our stored serum bank.
Immunizations
Bulk quantities of the PPPGMRPP peptide, a major antigenic region of the Sm B/B' autoantigen (6), and PSQQVMTP, a nonantigenic region of Sm B/B' beginning at amino acid 137 (for use as a negative control), were constructed on a polylysine backbone (MAP, Applied Biosystems, Foster City, CA), as suggested (13). Peptides constructed in this fashion will be designated with a -MAP suffix.
On day 1, CFA was emulsified with an equal volume (0.1 ml/0.1 ml) of sterile saline containing 100 µg of immunogen (peptide or control) and injected in equal portions i.p. and s.c. Boosting with 0.1 mg of immunogen in IFA into the s.c. tissue and i.p. space was conducted on days 10, 24, and 86. In addition, mice were serially bled on approximately days 17, 31, 49, and 105.
Mice from each strain were immunized with PPPGMRPP-MAP (experimental), PSQQVMTP-MAP (negative control), or Freund’s adjuvant without peptide.
ELISAs
Standard solid phase assays were used to measure the Ab
reactivity in mouse sera, as described in detail previously (5). One
microgram of Ag (Sm, nRNP, PPPGMRPP-MAP, PSQQVMTP-MAP, or Ro/SSA) was
coated per well in each of 96 polystyrene wells/plate. Mouse, human, or
rabbit sera, at varying dilutions, were added to each well and
incubated for 2 h at room temperature. After incubation, plates
were washed and incubated with anti-mouse, anti-human, or
anti-rabbit 
-chain-specific IgG raised in a goat, affinity
purified, and conjugated to alkaline phosphatase (Jackson
ImmunoResearch Laboratories (West Grove, PA) or Sigma Chemical Co. (St.
Louis, MO)) at a 1/10,000 or a 1/1,000 dilution, respectively.
Confirmatory ELISAs were performed as outlined above, with substitution
of rabbit anti-mouse 
-chain-specific Ig, affinity purified and
conjugated to alkaline phosphatase (Harlan Sera-Lab, Crawley Down, RI).
Para-nitrophenyl phosphate disodium was used as a substrate for
alkaline phosphatase, and plates were read at 405 nm with a micro-ELISA
reader (Dynatech, Alexandria, VA).
Solid phase peptide synthesis and Ab assay
The 1202 possible overlapping octapeptides of the spliceosome, Sm B/B', Sm D, nRNP 70K, nRNP A, and nRNP C, were prepared using solid phase chemistry as previously described (6, 14, 15, 16).
Wash steps and incubations were conducted in sealed plastic containers.
Other assay steps were performed by lowering the pins into microtiter
plate wells. First, pins were blocked with 3% low fat milk in PBS for
1 h at room temperature. Pins were then incubated in 1/100
dilutions of serum (mouse, rabbit, or human) in 3% milk/PBS with
0.05% Tween overnight at 4°C in humidified and sealed containers.
The pin blocks were then washed four times with 3% milk/PBS with
0.05% Tween for 10 min each time with vigorous agitation. Next, each
pin was incubated with anti-mouse or anti-human
-chain-specific IgG raised in a goat, affinity purified and
conjugated to alkaline phosphatase (Jackson ImmunoResearch
Laboratories) at a 1/10,000 dilution. Para-nitrophenyl phosphate
disodium was used as a substrate for alkaline phosphatase, and plates
were read at 405 nm with a micro-ELISA reader. Results for each plate
were then standardized by comparison with positive control pins. The
same peptide sequences were used as controls for all plates and were
allowed to develop to a specific absorbance with a known concentration
of a standard control serum. After completion of an assay, pins were
sonicated and washed to remove Abs, conjugate, and substrate (6).
Immunofluorescence
Mouse and human sera were tested for antinuclear Abs by a standard ANA test (INOVA Diagnostics, Inc., San Diego, CA) and for autoantibody binding to native DNA by an anti-nDNA test (Protrac Industries, Kerrville, TX), using previously described protocols (17, 18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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);
however, the strength and the timing of the response differed based
upon the strain immunized.
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A). Eighty
percent of the BALB/c mice also were early binders; however, due to the
inconsistent nature of their reactivity, they were not included in this
group. Sixty percent of the C3H/HeJ experimental animals immunized with PPPGMRPP-MAP died immediately after the second peptide immunization. Pathologic analysis of these animals showed signs of generalized vasodilitation, pulmonary hemorrhage, and other features consistent with anaphylaxis. No evidence of infection was found in these animals. No C3H/HeJ mice immunized with a negative control peptide or Freund’s alone had any sign of this type of reaction or of any other untoward consequence. The survival and apparent well-being of the control C3H/HeJ animals stand in stark contrast to the fatal reaction in the experimental animals.
All other strains of mice tested also developed Abs to the peptide of
immunization. SWR/J mice mounted varying responses, some of which were
equivalent to the earlier responders at 8 wk after the initial
immunization. NZB/Binj, C57BL/6J, C57L/J, C57BL/10J, and DBA/2J mice
all had mounted an immune response to PPPGMRPP by 8 wk after the
initial immunization (Fig. 1B). Several strains were
also tested using a -chain-specific conjugate to confirm these
findings (A/J, AKR/J, C57BL/6J, C57BL/J, BALB/c, PL/J, SWR/J, and
DBA/2J). Qualitative results for all strains were similar with both
conjugates, in that the ranks of the responses were identical. Among
all strains tested, BALB/c and DBA/2J titers appeared to be slightly
higher for the -chain-specific conjugate relative to the other
strains compared with the binding revealed for the whole IgG conjugate.
Also, SWR/J serum titers were slightly and relatively lower than those
in the other strains compared with findings using the whole IgG
conjugate. Interpretation of the data was the same, however, using
either conjugate.
In addition to binding the peptide of immunization, several strains of
mice developed Abs against the whole nRNP protein. The A/J, AKR/J,
129/J, and SJL/J mice all developed strong Ab titers to the whole Sm
and nRNP proteins. An example of SJL/J binding to PPPGMRPP-MAP is
presented in Figure 2. The A/J, 129/J,
and AKR/J anti-nRNP levels were similar to those presented for
SJL/J (Fig. 2). In addition, some, but not all, PPPGMRPP-MAP-immunized
BALB/c Bailey and PL/J mice developed significant, but lower, levels of
anti-nRNP Abs. The other inbred strains of mice tested did not
demonstrate binding to the Sm and nRNP proteins after PPPGMRPP-MAP
immunization.
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). These low levels of nRNP binding
(
0.1 OD) developed in animals immunized with the control peptide as
well as in those immunized with the experimental peptide. Even the
nonimmune animal had some minimal binding to nRNP at exsanguination.
These results show no evidence for an enduring anti-nRNP induced by
PPPGMRPP-MAP immunization in C57BL/J mice.
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, A and B).
These regions are antigenic in the MRL lpr/lpr spontaneous
animal model of SLE (15), the rabbit model of peptide-induced lupus
autoimmunity (5), and the human disease (6).
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, C and
D. At 28 days after immunization with PPPGMRPP-MAP, this
strain bound only the peptide of immunization. There was no evidence of
B cell epitope spreading, even at exsanguination. These results are
consistent with the failure of these same sera to bind to nRNP at this
time after immunization.
Finally, early experiments show that the SJL/J, A/J, 129/J, and AKR/J
peptide-immunized mice develop positive ANAs at titers of at least 1/80
(Fig. 5A), and the
AKR/J mice had titers >1/360. None of the preimmunization bleeds,
negative peptide control immunized mice, or Freund’s-immunized mice
developed anti-nuclear Abs (Fig. 5B).
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C. No other strains of mice immunized with
PPPGMRPP-MAP developed anti-dsDNA Abs. Preimmunization sera from
A/J and AKR/J mice as well as sham and negative control
peptide-immunized animals had no anti-dsDNA Abs.
Mice immunized with PPPGMRPP-MAP did not bind a negative control
peptide of Sm B/B' constructed on a MAP backbone or another lupus
autoantigen, Ro/SSA. In addition, preimmunization bleeds of these
animals did not bind the PPPGMRPP peptide in ELISA. Negative control
peptide-immunized animals as well as Freund’s-immunized animals did
not bind the PPPGMRPP sequence or the nRNP autoantigen. Representative
results of these negative assays are presented in Figures 2 and 3 (see
S6–S9 and L6–L10).
The H-2 region has been suspected to be critically important in this response. Fortunately, H-2 congenic strains are available with which to test this prediction. We immunized mouse strains congenic at the H-2 locus to explore the role of this region in susceptibility to peptide-induced lupus autoimmunity. The B6.AKR-H-2k/FlaEg with a B6 background and H-2 locus from AKR/J was immunized as described above. The B6 strain of mice mounts an immune response limited to the peptide of immunization, with no evidence of epitope spreading. These mice also do not mount an autoimmune response against whole Sm or nRNP Ag, nor did they produce anti-nuclear or anti-dsDNA Abs (data not presented). Replacing the H-2 locus of the nonresponder B6 strain with an H-2 locus from the responder AKR/J strain did not alter the resistance of the B6 mouse to peptide-induced lupus autoimmunity with PPPGMRPP-MAP.
The A.BY/Sn congenic strain, on the other hand, has the background
genes of the responder A/J strain with the congenic H-2 region of the
nonresponder C57BL/10J strain. These mice mounted a strong immune
response to the peptide of immunization and developed Abs that bound to
whole Sm and nRNP. This A/J congenic strain also developed Abs to
various other regions of Sm B/B' and Sm D and developed
anti-nuclear Abs. Based upon these data and those from the three
H-2k strains of mice, two of which mounted an expanded
response against the Sm and nRNP proteins (129/J and AKR/J) and another
of which did not (C3H/HeJ; Fig. 6), we
have no evidence that susceptibility to peptide-induced lupus
autoimmunity is mediated by H-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Particular Abs, here anti-PPPGMRPP, as well as the B cells that produce them are therefore likely to be important in the induction of lupus autoimmunity. Indeed, these findings strongly support a recently advocated concept postulating a central role for B cells in generating autoimmune responses (5, 20, 21). In our experiments, the B cell that binds both PPPGMRPP and endogenous spliceosomes of the mouse (which also would contain the peptide immunogen) is postulated to have the capacity not only to bind the spliceosome but also to process and present this and other spliceosome-derived peptides to T cells. With the appropriate immune stimulus, Ig epitope spreading and autoimmunity evidently follow. Existing data do not, however, distinguish between a central role of autoantigen processing by B cells and that of dendritic and other cells that are theoretically capable of this obviously required step in this process.
Early in these experiments we hypothesized that the strains that showed strong, early binding to the peptide of immunization would be the responder strains and that strains that mounted a late response would be nonresponders. However, several exceptions disprove this expected result. For example, significant overlap occurs between the degree of binding of responder strains and nonresponder strains to the peptide of immunization. In addition, we have two strains (129/J and BALB/c) that both have lower anti-PPPGMRPP titers (ranked 11 and 7, respectively, of 13 strains) and that bind whole nRNP and other commonly antigenic regions of the spliceosome and are therefore responder strains. The SWR/J strain has a very strong Ab response to PPPGMRPP (ranked fourth), yet never shows evidence of epitope spreading and is a nonresponder. Finally, directly comparing the anti-peptide immunization titers of various strains with different H-2 classes may be suboptimal. Based upon these data we suspect that the degree of anti-PPPGMRPP binding alone cannot predict responder phenotype.
Based upon other experimental animal models of lupus we fully expected H-2 immune response genes to play a major role in the development of anti-spliceosomal autoantibodies in the susceptible mouse strains. However, based upon the immune profiles showing responders and nonresponders with the same H-2 as well as the H-2 congenic experiments, our data unexpectedly suggest that H-2 of these mouse strains does not have a deciding role in the development of this autoimmune response. Many different genes are clearly important in natural immunity. For example, the gene found to be involved in resistance to tuberculosis as well as other intracellular pathogens is not associated with H-2 but is found on mouse chromosome 1, Nramp1 (natural resistance-associated macrophage protein-1) (22, 23).
Interesting work with the murine strains predisposed to spontaneous lupus show how complex the genetics of autoimmunity may be. For example, fas is the main gene responsible for lupus in the MRL lpr/lpr system (24), although a number of background genes also contribute. Yaa is important in the BSXB model (25, 26). Evaluation of the most classic animal model of SLE, NZBWF1 (or its derivatives), has shown that autoantibody production is related to a locus on chromosome 1 (27, 28, 29, 30, 31). Other loci important in this model include effects found on chromosomes 4, 11, and 17 (27, 28, 29, 30, 31). Whether this new model of autoimmunity described herein will have genetic effects at these same loci must await additional experiments. This model is fundamentally different from the spontaneous murine models of lupus, in that lupus autoimmunity is being induced in normal strains not otherwise known to develop lupus autoimmunity. Since induction of lupus autoimmunity is probably complex, requiring the participation of many components of the immune response, one could argue that the genes involved are probably different.
From another perspective, the extent to which clinical manifestations observed in our PPPGMRPP-MAP-immunized animals are directly attributable to and specific for an SLE autoimmune process is not known. The PPPGMRPP-MAP-immunized mice have variably had proteinuria, thrombocytopenia, and alopecia, which are all known to be part of human SLE (data not presented). Based upon the clinical and serologic similarities between this animal model and human disease, this model may serve an important role in dissecting the pathogenic mechanisms involved.
This murine model of peptide-induced lupus autoimmunity confirms and extends a new method of generating autoimmunity. This animal model presents a way to evaluate B cell epitope spreading that includes the observation of subsequent maturation of the autoimmune response (5). This model provides an important resource with which to develop an understanding of the steps in the pathophysiology of lupus autoimmunity. Finally, the strain variation observed and the absence of an H-2 effect on this process powerfully suggest that genes other than those at the MHC are important in this model.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Judith A. James, Arthritis and Immunology, Oklahoma Medical Research Foundation, 825 N.E. 13th St., Oklahoma City, OK 73104. E-mail address:
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; nRNP, nuclear ribonucleoprotein; NZB, New Zealand Black; ANA, anti-nuclear Abs.
Received for publication April 17, 1997. Accepted for publication September 15, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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