Clinical Immunology Newsletter Vol. 5, No. 2 February 1984

Single Gene Mutations that Cause SLE-Like Autoimmune Disease in Mice

Wendy F. Davidson, Ph.D. John B. Roths, B.A., and Herbert C. Morse III, M.D. Laboratory of Viral Diseases, National

Institute of Allergy and Infectious Diseases

National Institutes of Health Bethesda, Maryland The Jackson Laboratory Bar Harbor, Mahze

In the 88 years since the first com- prehensive description of systemic lupus erythematosus (SLE) by Osier (20), much progress has been made in determining the immunologic defects and pathogenic mechanisms of this spontaneous generalized autoimmune disease in man. Despite this progress, the etiology of SLE remains elusive. In an effort to gain a better under- standing of the pathogenesis of SLE in humans, in 1959, investigators turned to the study of inbred New Zealand (NZ) mice that spontaneously develop autoimmune disease. The two models utilized were NZB mice and (NZB × NZW)F l mice. NZB mice die prema- turely with hepatosplenomegaly and autoimmune hemolytic anemia, whereas, the hybrid mice develop a disease more closely resembling human SLE characterized by the ap- pearance of antinuclear antibodies and early-onset severe glomerulonephritis (2, I I). Although 20 years of intense investigation has not revealed the etiology of SLE in NZ mice, it has been established that these mice suffer from a wide range of immunologic ab- normalities, including intrinsic poly- clonal activation of B cells (9, 12, 14,

16, 28-30). Further evidence for the immunologic complexity of murine lupus has been provided by more re- cent studies of other SLE models, such as PN (7, 31), MRL, and BXSB (18, 28, 30). The inheritance of lupus in humans also appears to be complex. Studies of NZB mice crossed with nonautoimmune strains and recombi- nant inbred (RI) lines involving NZB have clearly demonstrated that there is no common genetic basis for all of the observed lymphocyte dysfunctions of NZB mice (3, 5, 8, 9, 14, 23, 32). Multiple independently assorting genes appeared to be responsible for poly- clonal B-cell activation, autoantibody formation, and T cell abnormalities (3, 5, 8, 9, 14, 23, 32). The exact enu- meration and identification of the genes responsible for disease in NZB hybrids and RI lines is complicated by the contribution of genes from the nonautoimmune parent that may alter- natively suppress or enhance the expression of NZB traits (5, 9, 32). Clearly, the NZB mouse is not an ideal model for determining how indi- vidual genes contribute to the autoim- mune process.

Research into the effects of single genes on autoimmune disease has been facilitated by the recent development of mice bearing single mutant genes that either induce or accelerate disease. Four genes are presently available for study, which include: (a) moth-eaten (me), an autosomal recessive mutant gene mapping to chromosome #6 (27). In C57BL/6 (B6) mice, me causes very severe rapid-onset autoimmune

disease and immunodeficiency with death occurring at 3 -4 weeks-of-age from autoimmune pneumonitis (I0, 27); (b) the Y-chromosome-linked au-

In This Issue

Single Gene Mutations that Cause SLE-Like Autoimmune Disease in Mice . . . . . . . . . . . . . . . . . . . 17 Experimental model to determine how SLE is hzduced at the molecular level

Autoantibody-Mediated Alteration of Cellular Adhesion: The Role of Plasminogen Activator . . . . . . . . . . . . . . . . . . . . . . . . . 20 Study of molecular mechanisms in pemphigus vulgaris and pemphigus foliaceous

Radioimmunoassay . . . . . . . . . . . . . . . 23 Discussion of its role hz clhffcal medichze

Government Forum . . . . . . . . . . . . . . 27 NCCLS documents for the clhffcal hnmunoIogy laboratory

Meeting Announcement . . . . . . . . . . 27

IUIS Report . . . . . . . . . . . . . . . . . . . . . . 28 Report of the Clhffcal hmnuno!ogy Committee

Meeting Report . . . . . . . . . . . . . . . . . . . 30 Second Annual Medical Laboratory Immunology Symposium

, | q i 1 | i i i

Table 1 Histologic and Serologic Characteristics of SLE Mice

Histologic Abnormalities

anti-

Lymphoid RB C

Strain Hyperplasia IC-GN Arteritis Arthritis MP NTA Ab

Serologic Abnormalities b

Anti-ss DNA

Anti-ds DNA Anti-

Anti-Nuclear SM

Ab Ab

Rheumatoid

Factor

gp70

anti-gp70

Complexes

N Z B + ( 2 - 3 x ) + - - + + + + -

( N Z B x N Z W ) F z + + + + - - + + + + -

B X S B tr 3 + ( 1 0 - 2 0 x ) + + + - - + -'- --- + -

MRL-Ipr/Ipr 4 + ( 1 0 0 x ) + + + + + + - --- + +

C3H.gld/gld 4 + (1 O0 x ) + - - - + NT + NT

B6-melme + + + - - N T + - - NT

Palmers ton

North (PN) + - 3 + + + + + - N T + __. + N T

D

m

+

N T

N T

+

+

+

+

N T

N T

N T

a MI, myocardial infarction. b Antibodies (Ab) to thymocytes (NTA), erythrocytes (RBC), single-stranded (ss) and double-stranded (ds) DNA and the nuclear glycoprot¢in, SM.

toimmune acceleration factor (Yaa) of strain BXSB/Mp. Males of this strain develop massive lymphadenopathy, hypergammaglobulinemia and autoanti- bodies, and die of acute immune-com- plex glomerulonephritis (IC-GN) at 5 - 6-months-of-age (17, 19, 30). These mice have been proposed as a model for human immunoblastic lymphade- nopathy (17). Compared with male mice, female BXSB mice develop late- onset chronic autoimmune disease and die at 15-16-months-of-age (17, 19, 30). The Yaa gene accelerates disease in a number of strains of mice predis- posed to late onset lupus, but has no effect on normal strains (19, 30); (c) lymphoproliferation (lpr), an un- mapped autosomal recessive gene with strain-dependent effects (17, 18); and (d) generalized lymphoproliferative disease (gld), a new autosomal reces- sive gene that maps to chromosome #1 and is not allelic with lpr (17, 24). Only Ipr and gld will be considered further in this review. The histologic and serologic characteristics of all SLE mice presently available for study are summarized in Table 1.

When introduced into mice that nor- mally develop delayed-onset, chronic autoimmune disease, such as MRL or NZB, Ipr causes early-onset massive lymphadenopathy (up to 100-fold en- largement), hypergammaglobulinemia, autoantibody production, and prema- ture death at 5-6-months-of-age (17, 18, 30; Izui et al., submitted). MRL- Iprllpr mice uniquely develop acute

polyarteritis, rheumatoid arthritis, and severe IC-GN (17, 18, 30). Similar pathology is not seen in NZB-Ipr/lpr mice, and the cause of death in this strain is unknown. In the context of these two strains, lpr can be viewed as a gene capable of accelerating and re- directing the process of autoimmunity.

By comparison, strains of mice that are not predisposed to autoimmune disease, such as SJL, AKR, C3H, and B6 are variably affected by lpr. Al- though lpr induces significant lympho- proliferation in each of these strains, this abnormality is variably expressed. Mice homozygous for lpr can be ranked in terms of diminishing degree of lymphoproliferation, as follows: SJL, MRL, NZB, C3H, AKR, and B6; and increasing longevity: SJL, MRL, NZB, AKR, B6, C3H (17, 25). All lpr homozygotes produce signifi- cant levels of antinuclear antibodies and anti-ds DNA antibodies (Izui et al., submitted). The cause of prema- ture death in SJL-, AKR-, C3H-, and B6-1pr homozygotes has not been es- tablished, however, none seem to suffer from life threatening IC-GN (25; J. B. Roths, unpublished observa- tions). By studying lpr on a number of different backgrounds, it is possible to begin to separate primary gene ef- fects from background modifications. Regardless of the strain into which it is introduced, lpr causes significant lymphadenopathy, the production of autoantibodies and premature death.

The newly discovered mutant gene,

gld, is very similar to lpr. C3H mice homozygous for gld develop early massive lymphodenopathy, hypergam- maglobulinemia, and autoantibodies to DNA (24). Like C3H-Ipr/Ipr mice, C3H-gld/gld mice only live half as long as normal controls (12-14 months), do not suffer from arthritis or arteritis, and rarely develop lupus-like nephritis (24). The cause of death in C3H-gld/gld mice appears to be inter- stitial pneumonitis without major peri- vascular lymphoid infiltrations (24). These mice, therefore, may prove to be a useful model for pulmonary dis- eases in humans, such as lymphocytic interstitial pneumonitis, idiopathic in- terstitial pneumonitis with immune complexes, and lupus pneumonitis (24).

A recent comparative study of lym- phoid abnormalities in C3H-lpr/lpr and C3H-gld/gld mice revealed many simi- larities of immune dysfunction. Phe- notypic studies of lymph node (LN) cells have shown that the characteristic lymphadenopathy of all strains of mice homozygous for Ipr and C3H-gld/gld mice seems to result from the expan- sion of an abnormal subset of T lym- phocytes. These T cells have mark- edly reduced levels of expression of Thy-1 antigen and Ly-I antigen, or are Thy- l - , and do not express Ly-2 or the B-cell markers, Ig, I-A, or ThB (13, 15). Surprisingly, there is also expression of Ly-5(B220), a member of the Ly-5 family of glycoproteins that is normally expressed only on B cells and pre-B cells (15). Despite

1 8 Cl in ica l Immunology News le t t e r

their expression of Ly-5(B220), these cells are thought to be of the T cell lineage, as they do not have Ig heavy chain gene rearrangements (15). Ly- 5(B220) + T cells initially are detected at 4-6-weeks-of-age, and by 10-12- weeks-of-age represent the predomi- nant population in the lymph nodes of lpr- and g/d-homozygotes. A partial replacement of the normal T-cell popu- lation in the spleen by Ly-5(B220) + T cells also is observed in these mice (15). The significance of expressing Ly-5(B220) on T cells is not known. Although no counterpart for Ly- 5(B220) + T cells has been found in normal mice, it is possible that these cells normally represent a very minor subpopulation of cells that is abnor- mally expanded in mice homozygous for lpr and gld. Alternatively, the expression of Ly-5(B220) on T cells may be truly aberrant and, eventually, may provide clues as to how the prod- ucts of lpr and gld genes function.

Generalized B cell hyperactivity is a cardinal feature of murine SLE (9, 12, 14, 16). C3H-gld/gld mice and all strains of mice homozygous for Ipr also develop early-onset polyclonal B cell activation evidenced by increased numbers of Ig-containing and Ig-se- creting cells, hypergammaglobulin- emia, and production of multiple au- toantibodies (12, 17, 24, Davidson et al., submitted). Studies of C3H mice homozygous for Ipr or gld have shown that B cells do not become activated until significant numbers of Ly-5 (B220) + T cells are present in spleen and LN (Davidson et al., submitted). This f'mding suggests that B-cell activa- tion may be secondary to T-cell abnor- malities rather than intrinsic, as is thought to be the case in NZB and (NZB × NZW) F 1 mice (4, 14, 16). Secondary activation of B cells by T- cell factors has been reported for MRL-Ipr/lpr mice (22, 24).

Another feature common to all mu- fine lupus strains is an age-related de- crease in the ability to produce inter- leukin 2 (IL-2) in vitro (30-32, Dav- idson et al., submitted). C3H-lpr/lpr and C3H-gld/gld mice also exhibit this abnormality (Davidson et al., in prepa- ration).

Both polyclonal B-cell activation (9,

12, 14, 16, 30) and defective IL-2 production (1, 6, 33) are thought to be the prodromes of early-onset, severe SLE with IC-GN. It should be stressed, however, that while C3H-, AKR-, and B6-1pr homozygotes ex- press both immunologic abnormalities, they do not develop this form of dis- ease. Clearly, host factors, in addition to B cell hyperactivity and depressed IL-2 production, are essential for the expression of fulminant SLE.

In summary, two independently as- sorting genes, lpr and gld, cause ap- parently similar abnormalities of the immune system. In at least one strain of mice (C3H), however, the diseases induced by these genes are very dif- ferent. These studies clearly demon- strate that, even with the simplified situation of single-gene models, the in- duction of autoimmune disease is a very complex process.

To conclude, murine models for SLE have provided investigators with a wealth of knowledge on the immune abnormalities expressed in autoimmune disease, and have offered insight into the complex genetic control of SLE. Despite this progress, very little is presently known about the triggering mechanisms in the autoimmune pro- cess. Hopefully, single-gene models of murine SLE will be useful tools for determining how SLE ultimately is in- duced at the molecular level.

This paper is dedicated to the late Dr. Edwin D. Murphy, who developed the MRL-Iprllpr and BXSB strains of mice that have become the models of choice for SLE in humans.

References 1. Altman, A. et al. (1981). Analysis of

T cell function in autoimmune murine strains. Defects in production of and responsiveness to interleukin 2. J. Exp. Med. 154:791-807.

2. Bielschowsky, M., B. J. ltelyer, and J. B. Howie. (1959). Spontaneous haemolytie anaemia in mice of the NZB/BL strain. Proc. Univ. Otago Med. Sch. 37:9-11.

3. Bocchieri, M. H. et al. (1982). Inde- pendent segregation of NZB autoim- mune abnormalities in (NZB x C58) recombinant inbred mice. Eur. J. Im- munol. 12:349-354.

4. Chused, T. M. et al. (1978). Mecha- nism of autoimmune disease in New Zealand black mice. In: N. R. Rose, P. E. Bigazzi, and N. L. Warner (eds), Genetic Control of Autoimmune Disease, Elsevier/North Holland, New York, pp. 177-190.

5. Datta, S. K. et al. (1982). Analysis of recombinant inbred lines derived from "autoimmune" (NZB) and "high leukemia" (C58) strains: Independent multigenic systems control B cell hy- peractivity, retrovirus expression, and autoimmunity. J. Immunol. 129:1539- 1544.

6. Dauphin~e, M. J. et al. (1981). In- terleukin 2 deficiency is a common feature of autoimmune mice. J. Im- munol. 127:2483-2487.

7. Davidson, W. F. (1982). Immuno- logic abnormalities of the autoimmune mouse, Palmerston North. J. Immunol. 129:751-758.

8. Davidson, W. F., T. M. Chused, and H. C. Morse, III. (1981). Ge- netic and functional analyses of the primary in vitro CTL response of NZB lymphocytes to H-2-compatible cells. Immunogenetics 12:445-463.

9. Davidson, W. F., T. M. Chused, and H. C. Morse, III. (1981). Ge- netic control of B - and T-lymphocyte abnormalities of NZB mice in crosses with B 10.D2 mice. Immunogenetics 13:421-434.

I0. Davidson, W. F. et al. (1979). Phe- notypic and functional effects of the motheaten gene on murine B and T lymphocytes. J. Immunol. 122:884- 891.

I 1. Howie, J. B. and B. J. Helyer. (1968). The immunology and pa- thology of NZB mice. Adv. Immunol. 9:215-266.

12. Izui, S., P. J. McConahey, and F. J. Dixon. (1978). Increased sponta- neous polyclonal activation of B lym- phocytes in mice with spontaneous au- toimmune disease. J. Immunol. 121:2213-2219.

13. Lewis, D. E., J. V. Giorgi, and N. L. Warner (1981). Flow cytometry analysis of T cells and continuous T cell lines from autoimmune MRL/I mice. Nature 289:298-300.

14. Manny, N., S. K. Datta, and R. S. Schwartz. (1979). Synthesis of IgM by cells of NZB and SWR mice and their crosses. J. Immunol. 122:1220- 1227.

15. Morse, H. C., III et al. (1982). Ab- normalities induced by the mutant gene lpr: Expansion of a unique lymphocyte subset. J. Immunol. 129:2612-2615.

16. Moutsopoulos, It. M. et al. (1977). Demonstration of activation of B lym- phocytes in New Zealand black mice

© 1984 by Elsevier Science Publishing Co.. Inc. 19

at birth by an immunoradiometric assay. J. lmmunol. 119:1639-1644.

17. Murphy, E. D. (1981). Lymphoproli- feration (Ipr) a mutant gene in strain MRL inducing murine lupus. In: M. E. Gershwin, and B. Merchant (eds.), bnmunologic Defects in Labo- ratory Animals, Vol. 2. Plenum New York, pp. 143-173.

18. Murphy, E. D. and J. B. Roths. (1979). Autoimmunity and lymphopro- liferation: Induction by mutant gene Ipr, and acceleration by a male-associ- ated factor in strain BXSB mice. In: N. R. Rose, P. E. Bigazzi, and N. L. Warner (eds.), Genetic Control of Au- toimmune Disease. Elsevier/North Hol- land, New York, pp. 207-220.

19. Murphy, E. D. and J. B. Roths. (1979). A Y-chromosome-associated factor producing accelerated autoim- munity and lymphoproliferation in strain BXSB. Arthrit. Rheum. 22:1188-1194.

20. Osier, W. (1895). On the visceral complications of erythema exudativum multiforme. J. Med. Sci. 110:629- 646.

21. Prud'Homme, G. et al. (1983). Iden- tification of a B cell differentiation

factor(s) spontaneously produced by proliferating T cells in murine lupus strains of the Ipr/Ipr genotype. J. Exp. Med. 157:730-742.

22. Prud'Homme, G. J. et al. (I983). 13 cell dependence on and response to ac- cessory signals in murine lupus strains. J. Exp. Med. 157:1815-1827.

23. Raveche, E. S. et al. (1981). Genetic studies in NZB mice. V. Recombinant inbred lines demonstrate that separate genes control autoimmune phenotype. J. Exp. Med. 153:1187-1197.

24. Roths, J. B., E. D. Murphy, and E. M. Eieher (1983). A new muta- tion, gld. producing lymphoprolifera- tion and autoimmunity in C3H/HeJ mice. J. Exp. Med. (in press).

25. Roths, J. B. et al. (1983). Modifica- tion of expression of lpr by back- ground genome. Fed. Proc. 42:1075.

26. Shultz, L. D., C. L. Bailey, and D. R. Coman. (1983). Haematopoietic stem cell function in motheaten mice. Exp. Hematol. 11:667-680.

27. Schultz, L. D. and M. C. Green. (1976). Motheaten, an immunodefi- cient mutant of the mouse. I1. De- pressed immune competence and ele-

vated serum immunoglobulin. J. lm- munol. 116:936-943.

28. Steinberg, A. D. et al. (1981). The cellular and genetic basis of murine lupus. Immunological Rev. 55:120- 154.

29. Talal, N. (1977). Autoimmunity and lymphoid malignancy: Manifestations of immunoregulatory disequilibrium. In: N. Talal (ed). Autoimmunity. Aca- demic Press, New York, pp. 184-206.

30. Theofilopoulos, A. N. and F. J. Dixon. (1981). Etiopathogenesis of murine SLE. Immunolog. Rev. 55:179-216.

31. Walker, S. E. et al. (1978). Palmer- ston North mice. a new animal model of systemic lupus erythemato~us. J. Lab. Clin. Med. 92:932-945.

32. Warner, N, L. (1977). Genetic as- pects of autoimmune disease in ani- mals. In: N. Talal (ed.), Autohn- munity, Genetic. Immunologic, Viro- logic, and Clinical Aspects. Academic Press, New York, pp. 33-62.

33. Wofsy, D. et al, (1981). Deficient in- terleukin 2 activity in MRL/Mp and C57BL/6J mice bearing the Ipr gene. J. Exp. Meal. 154:1671-1680.

Autoantibody-Mediated Alteration of Cellular Adhesion: The Role of Plasminogen Activator

Kay H. Singer, Ph.D. Division of Dermatology Duke University Medical Center Durham, North Carolina

Koji Hashimoto, M.D. , Ph.D. Department of Dermatology Osaka University Hospital Osaka, Japan

Gerald S. Lazarus, M.D. Department of Dermatology University of Pennsylvania Philadelphia, Pennsylvania

Adhesion is a basic attribute of many different cell types, necessary for a number o f cell functions, such as

organization of tissues and cell mo- tility. In addition, the adhesive prop- erties of some tumor cells are likely to be important in metastasis.

Alteration of cellular adhesive prop- erties can result in severe pathology. An example of this is the blistering au- toimmune disease, pemphigus, in which there is an alteration of cellular adhesion and, ultimately, a loss of cohesion of the epithelium of skin and mucous membranes. Biopsies of le- sional skin reveal rounding up of epi- dermal cells with loss o f epidermal cell adhesion; this pattern is referred to as acantholysis. Two major types of pemphigus are distinguished clinically and histopathologically. Pemphigus vulgaris exhibits extensive erosions, with vesicles forming just above the basal layer of epidermal cells. In con- trast, pemphigus foliaceous has very shallow blisters that appear in the more superficial granular layer of the

epidermis. We have investigated the mechanism for alteration of cellular adhesion in pemphigus at the molec- ular level, and present evidence that autoantibody stimulates the production of plasminogen activator and that plasmin is responsible for the loss of cellular adhesion (7).

The first clue to the etiology of pemphigus was established in 1964, when Beutner and Jordan (3) demon- strated that serum from pemphigus pa- tients contained autoantibodies that bound to an intercellular substance o f skin and mucosa. Subsequently, skin biopsies in vivo revealed deposition of autoantibodies in the epidermis of pemphigus patients. While these re- sults were intriguing, they did not prove that antibody played a role in the pathogenesis of the disease. Three lines of evidence now support a role for antibody in the pathogenesis of pemphigus: (a) correlation of antibody

20 Clinical Immunology Newsletter